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Optimization of a Precolumn OPA Derivatization HPLC Assay for Monitoring of l-Asparagine Depletion in Serum during l-Asparaginase Therapy

Optimization of a Precolumn OPA Derivatization HPLC Assay for Monitoring of l-Asparagine... Abstract A method for monitoring l-asparagine (ASN) depletion in patients’ serum using reversed-phase high-performance liquid chromatography with precolumn o-phthalaldehyde and ethanethiol (ET) derivatization is described. In order to improve the signal and stability of analytes, several important factors including precipitant reagent, derivatization conditions and detection wavelengths were optimized. The recovery of the analytes in biological matrix was the highest when 4% sulfosalicylic acid (1:1, v/v) was used as a precipitant reagent. Optimal fluorescence detection parameters were determined as λex = 340 nm and λem = 444 nm for maximal signal. The signal of analytes was the highest when the reagent ET and borate buffer of pH 9.9 were used in the derivatization solution. And the corresponding derivative products were stable up to 19 h. The validated method had been successfully applied to monitor ASN depletion and l-aspartic acid, l-glutamine, l-glutamic acid levels in pediatric patients during l-asparaginase therapy. Introduction l-Asparaginase (ASNase) is one of the essential drugs in the treatment of childhood acute lymphoblastic leukemia (ALL) clinically (1–3). ASNase catalyzes the deamination of l-asparagine (ASN) and l-glutamine (GLN) to l-aspartic acid (ASP) and l-glutamic acid (GLU), respectively (4–7). The anticancer activity of ASNase is believed to be associated primarily with the depletion of ASN, but the second glutaminase activity has also been implicated in its anticancer mechanism of action and may enhance cell death (2, 4). Reports have shown that most of the patients could achieve satisfactory therapeutic effects when the concentration of ASN in serum was kept below 3 nmol/mL (8–10). However, ASNase therapy is associated with a range of adverse effects, including normal organs and tissues impaired by excessive depletion of ASN and GLN and hypersensitivity reactions (7, 9, 11, 12). The pharmacokinetic, pharmacodynamic and immunogenic properties of different preparations are characterized by wide intra- and inter-individual variabilities (13). Therefore, it is very important to implement the therapeutic drug monitoring during treatment with ASNase. There have been a number of assay methods for the determination of amino acids in biological samples, which include liquid chromatography coupled with mass spectrometry analysis (LC–MS) (14–16), gas chromatography–mass spectrometry analysis (GC–MS) (17) and high-performance liquid chromatography (HPLC) methods coupled with fluorescence detector (FLD) (7, 18, 19). Most amino acids neither contain any strong chromophores nor fluorophores in their molecule and therefore a suitable derivatization method is necessary (20). One of the most well-characterized derivatization reagents used for the analysis of amino acid is o-phthalaldehyde (OPA) (21, 22). The use of OPA conjunction with sulfhydryl reagent could furnish fast reaction with primary amines (R2NH2) in alkaline solution at ambient temperature to form sulfonatoisoindoles that have high selectivity and sensitivity with ultraviolet detector (UVD) or FLD, and the OPA/sulfhydryl reagent itself does not interfere with the detection of amino acids (21, 23, 24). OPA-HPLC-FLD assay is one of the widely used methods for the current determination of amino acids (20, 24–26). However, the instability of isoindoles produced from amino acids and OPA, in the presence of different sulfhydryl compounds, as well as the instability of the OPA–sulfhydryl reagent itself is known as two of the main disadvantages of OPA derivatization reaction (23, 27). Further, both the efficiency of derivatization and the stability of derivative products are affected by the sulfhydryl reagent type, the ratio of amino acid to derivatization reagent and the buffer pH (21, 23, 28, 29). Successful quantitative endogenous analytes without a true blank matrix is a daunting challenge currently with a limited number of possible solutions. Blank biological matrix was prepared by adding ASNase to deplete endogenous ASN in serum in some methods (14, 19). However, the matrix contained more endogenous ASP and cannot be used to analyze the correlation between ASN and ASP during treatment with ASNase. In other methods, the standard working solutions were directly used for method validation (7, 30). Consequently, the aim of the present study was to establish a sensitive HPLC method for quantification of ASP, ASN, GLU and GLN in patients’ serum, which could enable implementation of therapeutic drug monitoring during treatment with ASNase. In order to improve chromatographic separation and signals of the analytes, the gradient elution program, protein precipitation, sulfhydryl reagent, buffer pH, excitation wavelength and emission wavelength were optimized thoroughly. Additionally, the biological matrix was obtained by treating the healthy human serum with powdered activated carbon to reduce the levels of endogenous analytes. After OPA derivatization, ASP and ASN were measured with the internal standard method by FLD, while GLU and GLN were measured with the external standard method by UVD. Experimental section Chemicals and reagents ASN, ASP, GLN, GLU and carbocisteine (internal standard, IS) were obtained from Chinese food and Drug Inspection Institute (Beijing, China). 2-mercaptoethanol (MCE), 3-mercaptopropionic acid (MPA), ethanethiol (ET), and 5-sulfosalicylic acid and OPA were purchased from Sigma-Aldrich (Shanghai, China). HPLC-grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). Sodium acetate anhydrous, boric acid, sodium hydroxide and potassium chloride were of analytical grade and purchased from Nanjing Chemical Reagent Co. Ltd. (Nanjing, China). Ultrapure water was prepared using Milli-Q Advantage A10 System (Merck Millipore, MA, USA). Liquid chromatography Liquid chromatography was performed with a Shimadzu LC-10AD HPLC system consisting of an SIL-HTc autosampler, an LC-10ADvp delivery system and a CTO-10Avp column oven. The analytical column was ZORBAX Eclipse AAA column (150 × 4.6 mm, 5 μm) fitted with the precolumn (12.5 × 4.6 mm, 5 μm) of the same type. The mobile phase was composed of sodium acetate (0.05 M; pH 7.2) (A) and sodium acetate (0.1 M)–acetonitrile–methanol (46:44:10, v/v/v) (pH 7.2) (B) under gradient elution condition: 26% B at 0.00–3.00 min, 26–51% B at 3.00–15.00 min, 51% B at 15.00–16.00 min, 100% B at 16.01–20.00 min, and 26% B at 20.01–23 min. The flow rate was 1 mL/min, the injection volume was 5 μL and the temperature of column was maintained at 40°C. FLD and UVD detections Optimal excitation and emission wavelengths were determined for ASP and ASN by performing excitation and emission scan using the 10AXL FLD. GLU and GLN were detected by the SPD-M10Avp UVD at 338 nm. Preparation of solutions Standard stock solutions of ASN (2.0 mg/mL) and GLN (5.0 mg/mL) were prepared in 50% methanol including 1% HCl. Standard stock solutions of ASP (2.0 mg/mL), GLU (3.0 mg/mL) and the IS (1.0 mg/mL) were prepared in 0.2 M HCl. Those stock solutions were diluted with water to prepare standard working solutions. All solutions were stored at 4°C. The borate buffer solution was consisted of 0.8 M boric acid (dissolved in 0.8 M KCl solution) and 0.8 M NaOH (1:1, v/v; pH 9.9 ± 0.05). The derivatization reagent was prepared by mixing 1 mL of OPA solution (10 mg/mL, dissolved in methanol), 2 mL borate buffer and 54 μL ET (OPA–ET), or 16 μL MCE (OPA–MCE), or 19 μL MPA (OPA–MPA). It is important to note that OPA–ET reagent needs to be freshly prepared every 2 days, OPA–MCE and OPA–MPA reagents are stable within 9 days (21). Sample preparation Collected blood samples were immediately cooled in an ice-water bath to decant the serum. The 50 μL of serum was deproteinized immediately with 50 μL of 4% sulfosalicylic acid solution. After vortex for 30 s, the mixture was centrifuged at 16000 rpm at 4°C for 10 min. The supernatant was stored at −80°C until analysis. Prior to analysis, it was thawed at room temperature, and then the mixture was vortexed and centrifuged for 10 min at 16000 rpm at 4°C. A 5 μL aliquot of the IS (50 μg/mL) was added to 50 μL of the supernatant, and 10 μL of the mixed solution was diluted by 40 μL mobile phase and alkalized with 1 M NaOH (1 μL 1% phenolphthalein solution was used as the indicator). ASP, ASN, GLU and GLN were determined by RP-HPLC after precolumn derivatization with OPA–sulfhydryl reagent. The three different reagents (acetonitrile, 4% sulfosalicylic acid and 8% sulfosalicylic acid) used for protein precipitation were tested and compared with each other to find the best opportunity to guarantee a high recovery. QC samples were prepared by adding mixed standard solutions to treated biological matrix. The treated biological matrix was obtained by centrifugation after the healthy human serum was incubated with powdered activated carbon overnight under gentle agitation. Derivatization procedure The derivatization was performed by mixing 50 μL of diluted and alkalized standard or sample solution with the derivatization reagent. The mixed solution was vortexed 30 s and analyzed by HPLC system. Several important factors, including the variety of sulfhydryl reagents (MCE, MPA and ET), the volume ratio of amino acid to derivatization reagent (5:1, 5:3, 5:5 and 5:10) and the pH of buffer (8.5–11), were tested to achieve the best results. The stability of derivative products was tested by comparing the signal intensity at different time points between 0.5 min and 19 h after adding the derivatization reagent. Method valuation The method was validated according to the bioanalytical method validation guidance currently accepted by the US Food and Drug Administration (US FDA) (31). Calibration standards were prepared by spiking 5 μL of appropriate working mixture solutions with 45 μL of treated biological matrix. After OPA derivatization, ASP (1.41–135.2 nmol/mL) and ASN (0.76–90.83 nmol/mL) were quantified using the internal standard method, so the peak area ratio of the analyte to that of IS was determined in both treated biological matrix and treated biological matrix + standard. The difference of these two ratios was used to construct the calibration curve. GLU (8.50–815.6 nmol/mL) and GLN (12.83–1232 nmol/mL) were quantified using the external standard method, so the peak area of the analyte which had been corrected by corresponding value in treated biological matrix was used to construct the calibration curve. The recovery exercise was performed at all QC levels by comparing the response of processed QC samples with those of the processed treated biological matrix added the same amount of analytes and IS standard solutions. The precision and the accuracy of the method were evaluated at all QC levels and these samples were analyzed in three consecutive batches on different days. Then the intra- and the inter-batch precision were expressed as the relative standard deviation (%RSD) of each sample’s measured concentration, and the accuracy was defined as a percentage of the measured concentration over the theoretical value. Stability of ASP, ASN, GLU and GLN in standard solutions and deproteinizad serum extracts were analyzed in triplicate for three QC levels. The stability of stock solution of analytes and IS was evaluated at 4°C for 10 days. Further, the stability of all working solutions were tested at room temperature for 8 h. The short-time stability of deproteinized serum extracts of QC samples were evaluated at room temperature for 8 h. The post-preparative stability was evaluated by reanalyzing the derivatized samples placed in the autosampler at 22°C for 10 h. The freeze and thaw stability were evaluated by analyzing deproteinized serum extracts after they had been frozen (−80°C) and thawed (room temperature) on 3 consecutive days. The long-time stability of deproteinized serum extracts was evaluated at −80°C for 15 days. Clinical application The established method was used to monitor ASN depletion and the concentration change of ASP, GLU and GLN in the serum of pediatric patients with ALL who were receiving polyethylene glycol (PEG) ASNase treatment. The children with ALL were treatment with five-drug combination chemotherapy (dexamethasone + daunorubicin + vincristine + 6-mercaptopurine + PEG ASNase). PEG ASNase at 2000 U/m2 was injected intramuscularly. Blood samples were collected at different times, namely, before administration, within 7 days and 7 days after administration. This study had been approved by the ethics committee. All blood samples were obtained under the condition that the parents of all children who were selected for this study were previously informed to obtain consent and provided written informed consent. Results Optimization of excitation and emission wavelengths The excitation and emission spectra show that the optimal excitation and emission wavelengths for ASP and ASN derivatives were around 340 and 444 nm, respectively. In addition, the chromatogram revealed a very good signal response and baseline stability at 340/444 nm. Optimization of OPA derivatization conditions The experimental results indicated that the products derived from each target amino acid with the three sulfhydryl reagents (MCE, MPA and ET) were extremely different in chromatographic retention behavior and detector response. Among those derivatives, the retention time of OPA–MPA products were shortest, while OPA–MCE products were eluted in much longer time. OPA–ET products had highest signal response (Figure 1). Figure 1. View largeDownload slide Effects of different sulfhydryl reagents on amino acid derivative signal. Figure 1. View largeDownload slide Effects of different sulfhydryl reagents on amino acid derivative signal. The effect of borate buffer pH ranging from 8.5 to 11 on the derivatization reaction was studied. Finally, it was found that the borate buffer with the pH 9.9 was the most helpful basic medium to derivatization. The volume ratio of amino acid to derivatization reagent was also optimized. The signal of derivative products reached the highest when the volume ratio of amino acid solution to OPA–ET was 5:3. When the waiting time was increased to 19 h after adding the derivatization reagent, the detector responses of the target analytes were unchanged. The results from our experiment indicated that OPA–ET derivatives were relatively stable during the waiting time from 0.5 min to 19 h (Figure 2). Figure 2. View largeDownload slide Stability of OPA–ET/amino acid derivatives within 19 h. Figure 2. View largeDownload slide Stability of OPA–ET/amino acid derivatives within 19 h. Optimization of extraction conditions of the serum It was found that the sulfosalicylic acid provided a 17–37% higher yield for the four amino acids, comparing to the recovery obtained with acetonitrile. Then, sulfosalicylic acid extraction conditions were further optimized by changing its concentration and adding volume. As shown in Figure 3, the best recovery was obtained with 4% sulfosalicylic acid when it was added at 1:1 ratio to the QC sample for all the analytes except ASP. Figure 3. View largeDownload slide Effects of different volumes and concentrations of sulfosalicylic acid on amino acid extraction. Figure 3. View largeDownload slide Effects of different volumes and concentrations of sulfosalicylic acid on amino acid extraction. Method valuation Under the established HPLC–UVD–FLD conditions, no obvious endogenous interfering peaks at the elution times of ASP (5.9 min), ASN (13.2 min), GLU (7.9 min), GLN (14.4 min) and IS (9.9 min) were detected in real samples. Figure 4 presents representative chromatograms for ASN, ASP, GLN, GLU and IS resulting from the untreated biological matrix (Figure 4A), treated biological matrix (Figure 4B), QC sample (Figure 4C) and patient serum (Figure 4D). Figure 4. View largeDownload slide Representative chromatograms of OPA derivatives. ASN and ASP chromatograms obtained from the untreated biological matrix (A1), treated biological matrix (B1), QC sample (C1) and patient serum (D1). GLN and GLU chromatograms obtained from the untreated biological matrix (A2), treated biological matrix (B2), QC sample (C2) and patient serum (D2). Figure 4. View largeDownload slide Representative chromatograms of OPA derivatives. ASN and ASP chromatograms obtained from the untreated biological matrix (A1), treated biological matrix (B1), QC sample (C1) and patient serum (D1). GLN and GLU chromatograms obtained from the untreated biological matrix (A2), treated biological matrix (B2), QC sample (C2) and patient serum (D2). The extraction efficiency of three QC levels were consistent, precise and reproducible. The mean extraction recoveries ranged from 105% to 109% for ASP, 105% to 109% for ASN, 95% to 100% for GLU and 93% to 99% for GLN. In the assay of serum ASN, ASP, GLN and GLU, all the weighted calibration curves showed excellent linearity within tested concentration ranges, with the correlation coefficients (r) greater than 0.999. The equations of ASN, ASP, GLN and GLU calibration curve were C = 35.71 A/AIS + 0.24 (0.76–90.83 nmol/mL), C = 88.37 A/AIS + 0.29 (1.42–135.2 nmol/mL), C = 9.17 × 10–3 A − 1.63 (12.83–1232 nmol/mL) and C = 9.11 × 10–3 A − 0.73 (8.50–815.6 nmol/mL), respectively. The lower limit of quantification (LLOQ) of ASN was 0.76 nmol/mL which was measured from five independent samples and the precision (RSD = 9.34%) and accuracy (85.50–113.3%) were acceptable. The accuracy and the intra- and the inter-batch precision of the method were expressed as a percentage of the measured concentration over the theoretical value and a %RSD, respectively. The results indicated that the accuracy and the intra- and inter-batch precision were within the defined acceptance criteria (31). The accuracy ranged from 98.82% to 110.1%, intra- and inter-batch precision were less than 12% (Table I). Table I. The Precision and Accuracy of the Assay for Determining ASN, ASP, GLN, GLU in the Serum Concentration added (nmol/mL) Intra-day (n = 5) Inter-day (n = 15) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) ASP 135.2 138.5 ± 2.21 1.60 102.42 138.7 ± 2.41 1.74 102.55 18.03 19.04 ± 0.50 2.64 105.60 18.99 ± 0.40 2.10 105.32 2.82 2.96 ± 0.30 10.18 105.23 2.88 ± 0.28 9.73 102.16 ASN 90.83 90.74 ± 2.11 2.32 99.91 92.42 ± 2.03 2.20 101.75 12.11 12.23 ± 0.14 1.16 101.01 12.50 ± 0.28 2.28 103.20 1.89 1.88 ± 0.14 7.28 99.32 1.97 ± 0.17 8.76 103.98 GLU 815.6 824.8 ± 18.81 2.28 101.13 799.7 ± 24.75 3.10 98.05 108.7 114.3 ± 1.53 1.34 105.15 113.4 ± 5.18 4.57 104.24 16.99 16.95 ± 1.93 11.36 99.75 16.92 ± 1.83 10.82 99.55 GLN 1232 1217 ± 33.93 2.79 98.82 1198 ± 30.73 2.56 97.29 164.2 171.5 ± 4.02 2.35 104.41 164.5 ± 6.39 3.88 100.15 25.66 28.23 ± 1.45 5.15 110.03 26.72 ± 1.89 7.06 104.14 Concentration added (nmol/mL) Intra-day (n = 5) Inter-day (n = 15) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) ASP 135.2 138.5 ± 2.21 1.60 102.42 138.7 ± 2.41 1.74 102.55 18.03 19.04 ± 0.50 2.64 105.60 18.99 ± 0.40 2.10 105.32 2.82 2.96 ± 0.30 10.18 105.23 2.88 ± 0.28 9.73 102.16 ASN 90.83 90.74 ± 2.11 2.32 99.91 92.42 ± 2.03 2.20 101.75 12.11 12.23 ± 0.14 1.16 101.01 12.50 ± 0.28 2.28 103.20 1.89 1.88 ± 0.14 7.28 99.32 1.97 ± 0.17 8.76 103.98 GLU 815.6 824.8 ± 18.81 2.28 101.13 799.7 ± 24.75 3.10 98.05 108.7 114.3 ± 1.53 1.34 105.15 113.4 ± 5.18 4.57 104.24 16.99 16.95 ± 1.93 11.36 99.75 16.92 ± 1.83 10.82 99.55 GLN 1232 1217 ± 33.93 2.79 98.82 1198 ± 30.73 2.56 97.29 164.2 171.5 ± 4.02 2.35 104.41 164.5 ± 6.39 3.88 100.15 25.66 28.23 ± 1.45 5.15 110.03 26.72 ± 1.89 7.06 104.14 Table I. The Precision and Accuracy of the Assay for Determining ASN, ASP, GLN, GLU in the Serum Concentration added (nmol/mL) Intra-day (n = 5) Inter-day (n = 15) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) ASP 135.2 138.5 ± 2.21 1.60 102.42 138.7 ± 2.41 1.74 102.55 18.03 19.04 ± 0.50 2.64 105.60 18.99 ± 0.40 2.10 105.32 2.82 2.96 ± 0.30 10.18 105.23 2.88 ± 0.28 9.73 102.16 ASN 90.83 90.74 ± 2.11 2.32 99.91 92.42 ± 2.03 2.20 101.75 12.11 12.23 ± 0.14 1.16 101.01 12.50 ± 0.28 2.28 103.20 1.89 1.88 ± 0.14 7.28 99.32 1.97 ± 0.17 8.76 103.98 GLU 815.6 824.8 ± 18.81 2.28 101.13 799.7 ± 24.75 3.10 98.05 108.7 114.3 ± 1.53 1.34 105.15 113.4 ± 5.18 4.57 104.24 16.99 16.95 ± 1.93 11.36 99.75 16.92 ± 1.83 10.82 99.55 GLN 1232 1217 ± 33.93 2.79 98.82 1198 ± 30.73 2.56 97.29 164.2 171.5 ± 4.02 2.35 104.41 164.5 ± 6.39 3.88 100.15 25.66 28.23 ± 1.45 5.15 110.03 26.72 ± 1.89 7.06 104.14 Concentration added (nmol/mL) Intra-day (n = 5) Inter-day (n = 15) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) ASP 135.2 138.5 ± 2.21 1.60 102.42 138.7 ± 2.41 1.74 102.55 18.03 19.04 ± 0.50 2.64 105.60 18.99 ± 0.40 2.10 105.32 2.82 2.96 ± 0.30 10.18 105.23 2.88 ± 0.28 9.73 102.16 ASN 90.83 90.74 ± 2.11 2.32 99.91 92.42 ± 2.03 2.20 101.75 12.11 12.23 ± 0.14 1.16 101.01 12.50 ± 0.28 2.28 103.20 1.89 1.88 ± 0.14 7.28 99.32 1.97 ± 0.17 8.76 103.98 GLU 815.6 824.8 ± 18.81 2.28 101.13 799.7 ± 24.75 3.10 98.05 108.7 114.3 ± 1.53 1.34 105.15 113.4 ± 5.18 4.57 104.24 16.99 16.95 ± 1.93 11.36 99.75 16.92 ± 1.83 10.82 99.55 GLN 1232 1217 ± 33.93 2.79 98.82 1198 ± 30.73 2.56 97.29 164.2 171.5 ± 4.02 2.35 104.41 164.5 ± 6.39 3.88 100.15 25.66 28.23 ± 1.45 5.15 110.03 26.72 ± 1.89 7.06 104.14 The results of stability tests at three QC levels (n = 3) are summarized in Table II. And the stability of the standard amino acid solutions are shown in Supplementary Table I. The data show reliable stability behavior of analytes and their derivative products under the condition tested. Table II. Stability of ASP, ASN, GLU, GLN in Human Serum at Different QC Levels (n = 3) Condition of sample analysis Concentration measured (mean ± SD) ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) 2.82 18.03 135.2 1.89 12.11 90.83 16.99 108.7 815.6 25.66 164.2 1232 Measured immediately (0 h) 3.22 ± 0.06 19.43 ± 0.16 138.0 ± 2.56 1.86 ± 0.15 12.43 ± 0.13 90.94 ± 2.09 15.31 ± 1.39 121.0 ± 2.78 773.3 ± 6.72 24.97 ± 4.02 163.8 ± 9.08 1261 ± 38.77 Short-term stability (8 h) 3.25 ± 0.03 19.33 ± 0.15 135.8 ± 1.64 1.91 ± 0.11 12.36 ± 0.14 89.03 ± 0.23 15.87 ± 1.37 123.1 ± 0.40 775.7 ± 3.00 28.96 ± 0.57 155.2 ± 1.87 1232 ± 26.79 Long-term stability (15 d) 3.00 ± 0.22 19.98 ± 0.47 146.1 ± 6.10 1.91 ± 0.17 12.85 ± 0.38 94.56 ± 1.99 15.26 ± 1.05 118.1 ± 3.70 765.4 ± 9.10 23.01 ± 2.35 176.5 ± 5.26 1258 ± 36.23 Post-preparative stability (10 h) 3.15 ± 0.10 17.92 ± 1.66 134.6 ± 5.67 1.76 ± 0.18 11.73 ± 0.80 88.60 ± 4.35 16.22 ± 1.85 111.1 ± 8.15 758.7 ± 15.16 23.84 ± 3.01 177.5 ± 5.72 1267 ± 32.69 Freeze and thaw stability (first time) 3.07 ± 0.22 18.98 ± 0.62 138.3 ± 2.12 1.90 ± 0.23 12.06 ± 0.47 87.55 ± 5.37 15.70 ± 1.36 120.0 ± 3.97 781.1 ± 13.31 22.40 ± 2.12 178.2 ± 6.37 1314 ± 30.84 Freeze and thaw stability (second time) 3.12 ± 0.13 18.98 ± 0.54 137.3 ± 3.27 2.00 ± 0.23 12.00 ± 0.50 87.71 ± 5.25 14.97 ± 0.86 119.1 ± 2.81 783.7 ± 18.74 22.15 ± 1.84 174.5 ± 4.57 1280 ± 63.94 Freeze and thaw stability (third time) 3.02 ± 0.19 18.53 ± 0.69 135.7 ± 3.44 2.03 ± 0.21 12.01 ± 0.50 87.69 ± 5.39 15.87 ± 1.29 118.7 ± 5.39 787.0 ± 15.49 22.96 ± 1.91 177.9 ± 5.20 1308 ± 53.91 Condition of sample analysis Concentration measured (mean ± SD) ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) 2.82 18.03 135.2 1.89 12.11 90.83 16.99 108.7 815.6 25.66 164.2 1232 Measured immediately (0 h) 3.22 ± 0.06 19.43 ± 0.16 138.0 ± 2.56 1.86 ± 0.15 12.43 ± 0.13 90.94 ± 2.09 15.31 ± 1.39 121.0 ± 2.78 773.3 ± 6.72 24.97 ± 4.02 163.8 ± 9.08 1261 ± 38.77 Short-term stability (8 h) 3.25 ± 0.03 19.33 ± 0.15 135.8 ± 1.64 1.91 ± 0.11 12.36 ± 0.14 89.03 ± 0.23 15.87 ± 1.37 123.1 ± 0.40 775.7 ± 3.00 28.96 ± 0.57 155.2 ± 1.87 1232 ± 26.79 Long-term stability (15 d) 3.00 ± 0.22 19.98 ± 0.47 146.1 ± 6.10 1.91 ± 0.17 12.85 ± 0.38 94.56 ± 1.99 15.26 ± 1.05 118.1 ± 3.70 765.4 ± 9.10 23.01 ± 2.35 176.5 ± 5.26 1258 ± 36.23 Post-preparative stability (10 h) 3.15 ± 0.10 17.92 ± 1.66 134.6 ± 5.67 1.76 ± 0.18 11.73 ± 0.80 88.60 ± 4.35 16.22 ± 1.85 111.1 ± 8.15 758.7 ± 15.16 23.84 ± 3.01 177.5 ± 5.72 1267 ± 32.69 Freeze and thaw stability (first time) 3.07 ± 0.22 18.98 ± 0.62 138.3 ± 2.12 1.90 ± 0.23 12.06 ± 0.47 87.55 ± 5.37 15.70 ± 1.36 120.0 ± 3.97 781.1 ± 13.31 22.40 ± 2.12 178.2 ± 6.37 1314 ± 30.84 Freeze and thaw stability (second time) 3.12 ± 0.13 18.98 ± 0.54 137.3 ± 3.27 2.00 ± 0.23 12.00 ± 0.50 87.71 ± 5.25 14.97 ± 0.86 119.1 ± 2.81 783.7 ± 18.74 22.15 ± 1.84 174.5 ± 4.57 1280 ± 63.94 Freeze and thaw stability (third time) 3.02 ± 0.19 18.53 ± 0.69 135.7 ± 3.44 2.03 ± 0.21 12.01 ± 0.50 87.69 ± 5.39 15.87 ± 1.29 118.7 ± 5.39 787.0 ± 15.49 22.96 ± 1.91 177.9 ± 5.20 1308 ± 53.91 Table II. Stability of ASP, ASN, GLU, GLN in Human Serum at Different QC Levels (n = 3) Condition of sample analysis Concentration measured (mean ± SD) ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) 2.82 18.03 135.2 1.89 12.11 90.83 16.99 108.7 815.6 25.66 164.2 1232 Measured immediately (0 h) 3.22 ± 0.06 19.43 ± 0.16 138.0 ± 2.56 1.86 ± 0.15 12.43 ± 0.13 90.94 ± 2.09 15.31 ± 1.39 121.0 ± 2.78 773.3 ± 6.72 24.97 ± 4.02 163.8 ± 9.08 1261 ± 38.77 Short-term stability (8 h) 3.25 ± 0.03 19.33 ± 0.15 135.8 ± 1.64 1.91 ± 0.11 12.36 ± 0.14 89.03 ± 0.23 15.87 ± 1.37 123.1 ± 0.40 775.7 ± 3.00 28.96 ± 0.57 155.2 ± 1.87 1232 ± 26.79 Long-term stability (15 d) 3.00 ± 0.22 19.98 ± 0.47 146.1 ± 6.10 1.91 ± 0.17 12.85 ± 0.38 94.56 ± 1.99 15.26 ± 1.05 118.1 ± 3.70 765.4 ± 9.10 23.01 ± 2.35 176.5 ± 5.26 1258 ± 36.23 Post-preparative stability (10 h) 3.15 ± 0.10 17.92 ± 1.66 134.6 ± 5.67 1.76 ± 0.18 11.73 ± 0.80 88.60 ± 4.35 16.22 ± 1.85 111.1 ± 8.15 758.7 ± 15.16 23.84 ± 3.01 177.5 ± 5.72 1267 ± 32.69 Freeze and thaw stability (first time) 3.07 ± 0.22 18.98 ± 0.62 138.3 ± 2.12 1.90 ± 0.23 12.06 ± 0.47 87.55 ± 5.37 15.70 ± 1.36 120.0 ± 3.97 781.1 ± 13.31 22.40 ± 2.12 178.2 ± 6.37 1314 ± 30.84 Freeze and thaw stability (second time) 3.12 ± 0.13 18.98 ± 0.54 137.3 ± 3.27 2.00 ± 0.23 12.00 ± 0.50 87.71 ± 5.25 14.97 ± 0.86 119.1 ± 2.81 783.7 ± 18.74 22.15 ± 1.84 174.5 ± 4.57 1280 ± 63.94 Freeze and thaw stability (third time) 3.02 ± 0.19 18.53 ± 0.69 135.7 ± 3.44 2.03 ± 0.21 12.01 ± 0.50 87.69 ± 5.39 15.87 ± 1.29 118.7 ± 5.39 787.0 ± 15.49 22.96 ± 1.91 177.9 ± 5.20 1308 ± 53.91 Condition of sample analysis Concentration measured (mean ± SD) ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) 2.82 18.03 135.2 1.89 12.11 90.83 16.99 108.7 815.6 25.66 164.2 1232 Measured immediately (0 h) 3.22 ± 0.06 19.43 ± 0.16 138.0 ± 2.56 1.86 ± 0.15 12.43 ± 0.13 90.94 ± 2.09 15.31 ± 1.39 121.0 ± 2.78 773.3 ± 6.72 24.97 ± 4.02 163.8 ± 9.08 1261 ± 38.77 Short-term stability (8 h) 3.25 ± 0.03 19.33 ± 0.15 135.8 ± 1.64 1.91 ± 0.11 12.36 ± 0.14 89.03 ± 0.23 15.87 ± 1.37 123.1 ± 0.40 775.7 ± 3.00 28.96 ± 0.57 155.2 ± 1.87 1232 ± 26.79 Long-term stability (15 d) 3.00 ± 0.22 19.98 ± 0.47 146.1 ± 6.10 1.91 ± 0.17 12.85 ± 0.38 94.56 ± 1.99 15.26 ± 1.05 118.1 ± 3.70 765.4 ± 9.10 23.01 ± 2.35 176.5 ± 5.26 1258 ± 36.23 Post-preparative stability (10 h) 3.15 ± 0.10 17.92 ± 1.66 134.6 ± 5.67 1.76 ± 0.18 11.73 ± 0.80 88.60 ± 4.35 16.22 ± 1.85 111.1 ± 8.15 758.7 ± 15.16 23.84 ± 3.01 177.5 ± 5.72 1267 ± 32.69 Freeze and thaw stability (first time) 3.07 ± 0.22 18.98 ± 0.62 138.3 ± 2.12 1.90 ± 0.23 12.06 ± 0.47 87.55 ± 5.37 15.70 ± 1.36 120.0 ± 3.97 781.1 ± 13.31 22.40 ± 2.12 178.2 ± 6.37 1314 ± 30.84 Freeze and thaw stability (second time) 3.12 ± 0.13 18.98 ± 0.54 137.3 ± 3.27 2.00 ± 0.23 12.00 ± 0.50 87.71 ± 5.25 14.97 ± 0.86 119.1 ± 2.81 783.7 ± 18.74 22.15 ± 1.84 174.5 ± 4.57 1280 ± 63.94 Freeze and thaw stability (third time) 3.02 ± 0.19 18.53 ± 0.69 135.7 ± 3.44 2.03 ± 0.21 12.01 ± 0.50 87.69 ± 5.39 15.87 ± 1.29 118.7 ± 5.39 787.0 ± 15.49 22.96 ± 1.91 177.9 ± 5.20 1308 ± 53.91 Clinical application Table III shows the level of ASN depletion and the change of concentration of ASP, GLU and GLN in pediatric patients before and after the course of therapy. Median ASN concentrations prior to treatment were 58.10 nmol/mL. After ASNase administration, ASN was consumed and its concentration was below the level of detection within 7 days, and since the 8th day, the ASN level began to rise and returned to the original state. In addition, after ASNase treatment, the concentration of GLN was significantly decreased, while the concentration of GLU was significantly increased, then the amino acids were also gradually returned to the original state from the 8th day. Table III. Monitoring ASN Depletion and the Concentration of ASP, GLU and GLN in ALL Children who Received 2000 U/m2 PEG Time of blood sampling ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) Median Median Median Median Range Range Range Range Prior treatment 62.01 58.10 154.1 367.3 (30.27–116.8) (29.14–141.3) (105.7–303.7) (187.7–455.2) (n = 9) (n = 9) (n = 9) (n = 9) Post-treatment (within 7 days) 64.62 N.D.a 384.7**b 136.4**b (23.96–86.19) (258.5–677.9) (22.23–204.2) (n = 12) (n = 12) (n = 12) (n = 12) Post-treatment (after 8 days) 46.02 69.01 163.3*c 301.2*c (33.95–67.85) (52.21–98.62) (49.44–174.7) (196.2–381.98) (n = 5) (n = 5) (n = 5) (n = 5) Time of blood sampling ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) Median Median Median Median Range Range Range Range Prior treatment 62.01 58.10 154.1 367.3 (30.27–116.8) (29.14–141.3) (105.7–303.7) (187.7–455.2) (n = 9) (n = 9) (n = 9) (n = 9) Post-treatment (within 7 days) 64.62 N.D.a 384.7**b 136.4**b (23.96–86.19) (258.5–677.9) (22.23–204.2) (n = 12) (n = 12) (n = 12) (n = 12) Post-treatment (after 8 days) 46.02 69.01 163.3*c 301.2*c (33.95–67.85) (52.21–98.62) (49.44–174.7) (196.2–381.98) (n = 5) (n = 5) (n = 5) (n = 5) aNot detection; *P < 0.01,**P < 0.001. bPrior treatment vs. post-treatment (within 7 days). cPost-treatment (within 7 days) vs. post-treatment (after 8 days). Table III. Monitoring ASN Depletion and the Concentration of ASP, GLU and GLN in ALL Children who Received 2000 U/m2 PEG Time of blood sampling ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) Median Median Median Median Range Range Range Range Prior treatment 62.01 58.10 154.1 367.3 (30.27–116.8) (29.14–141.3) (105.7–303.7) (187.7–455.2) (n = 9) (n = 9) (n = 9) (n = 9) Post-treatment (within 7 days) 64.62 N.D.a 384.7**b 136.4**b (23.96–86.19) (258.5–677.9) (22.23–204.2) (n = 12) (n = 12) (n = 12) (n = 12) Post-treatment (after 8 days) 46.02 69.01 163.3*c 301.2*c (33.95–67.85) (52.21–98.62) (49.44–174.7) (196.2–381.98) (n = 5) (n = 5) (n = 5) (n = 5) Time of blood sampling ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) Median Median Median Median Range Range Range Range Prior treatment 62.01 58.10 154.1 367.3 (30.27–116.8) (29.14–141.3) (105.7–303.7) (187.7–455.2) (n = 9) (n = 9) (n = 9) (n = 9) Post-treatment (within 7 days) 64.62 N.D.a 384.7**b 136.4**b (23.96–86.19) (258.5–677.9) (22.23–204.2) (n = 12) (n = 12) (n = 12) (n = 12) Post-treatment (after 8 days) 46.02 69.01 163.3*c 301.2*c (33.95–67.85) (52.21–98.62) (49.44–174.7) (196.2–381.98) (n = 5) (n = 5) (n = 5) (n = 5) aNot detection; *P < 0.01,**P < 0.001. bPrior treatment vs. post-treatment (within 7 days). cPost-treatment (within 7 days) vs. post-treatment (after 8 days). Discussion Most of the analytical chemists, who had worked or are working in the field of amino acids and tried to use various derivatization techniques in their chromatographic determination, know that the OPA precolumn derivatization is a rapid and accurate method to determinate the concentrations of amino acids (21, 23, 28, 29). However, the instability of amino acid derivatives has attracted much attention (23, 29). In the experiment, three kinds of sulfhydryl reagents were compared. Among them, the OPA–ET derivatives exhibit the highest fluorescence and stability within 19 h. Petra Drábková et al. (20) found that the OPA–MCE derivatives are only stable for about half an hour and then a 30% decrease in fluorescence intensity was observed. Ibolya Molnár-Perl et al. (23) reported that OPA–MPA and OPA-N-acetyl-L-cysteine (NAC) derivatives are stable within 6 h. The optimal excitation and emission wavelengths were determined to guarantee the most sensitive fluorescence signal to detect the OPA–ET derivatives of ASN, ASP, GLN and GLU. The elution condition was optimized to achieve reliable peak separation in human serum samples, further confirming no interfering peaks were present in biological samples under the optimized elution program. We validated the method by quantifying analyte concentration using internal or external standards. The healthy human serum which incubated with activated carbon to reduce the endogenous analytes was used as a biological matrix to ensure the quantitative accuracy (Figure 4). The ASN and ASP concentrations could be quantified satisfactorily with FLD detection. Meanwhile, the concentration of GLN and GLU in the sample exceeded the range of the detector generating a flat-topped peak. Thus, using both UVD and FLD enables simultaneous quantification of all analytes. The limit of detection of ASN is 0.76 nmol/mL, which is qualified to monitor the depletion of serum ASN. The current method is optimized with the elution time of approximately 15 min, and another flush was utilized to wash out late eluting peaks. The run time is totally about 23 min, which is shorter than 35 min in the method reported by Christa E. Nath et al. (7). The effectiveness of the proposed method for monitoring ASN depletion during ASNase treatment was verified by analyzing the serum samples from the pediatric patient. Since the aim of ASNase therapy is to completely deplete the extracellular ASN, ASN concentration will always be under the threshold of 3 nmol/mL and close to the LOQ of the method (7, 9). It is worth mentioning that our method for ASN determination has good reproducibility (<10%) even at the low concentration of 1.89 nmol/mL. ASNase administration succeeded in depleting ASN concentrations to 0.76 nmol/mL or lower, and the change trend of four amino acid was in agreement with reference (7, 32). In summary, we have developed a relatively novel precolumn OPA derivatization HPLC assay which was qualified for the therapeutic drug monitoring during treatment with ASNase. Acknowledgments We would like to thank the patients who took part in this study and the all co-workers in Children’s Hospital of Nanjing Medical University and China Pharmaceutical University. Funding This project was financially supported by Medical Science and technology development Foundation, Nanjing Department of Health (No. YKK14112), the National Natural Science Foundation of China (No. 81403314) and the Natural Research Foundation of Jiangsu Province of china (No. BK20161456). Conflict of interest statement The authors declare no conflict of interest. References 1 Pui , C.H. , Evans , W.E. ; Treatment of acute lymphoblastic leukemia ; The New England Journal of Medicine , ( 2006 ); 354 ( 2 ): 166 – 178 . Google Scholar Crossref Search ADS PubMed 2 Purwaha , P. , Lorenzi , P.L. , Silva , L.P. , Hawke , D.H. , Weinstein , J.N. ; Targeted metabolomic analysis of amino acid response to l-asparaginase in adherent cells ; Metabolomics: Official Journal of the Metabolomic Society , ( 2014 ); 10 ( 5 ): 909 – 919 . Google Scholar PubMed 3 Muller , H.J. , Boos , J. ; Use of l-asparaginase in childhood ALL ; Critical Reviews in Oncology/Hematology , ( 1998 ); 28 ( 2 ): 97 – 113 . Google Scholar Crossref Search ADS PubMed 4 Miller , H.K. , Salser , J.S. , Balis , M.E. ; Amino acid levels following l-asparagine amidohydrolase (EC.3.5.1.1) Therapy ; Cancer Research , ( 1969 ); 29 ( 1 ): 183 – 187 . Google Scholar PubMed 5 Kawedia , J.D. , Rytting , M.E. ; Asparaginase in acute lymphoblastic leukemia ; Clinical Lymphoma, Myeloma & Leukemia , ( 2014 ); 14 ( S3 ): S14 – S17 . Google Scholar Crossref Search ADS PubMed 6 Lopes , A.M. , Oliveira-Nascimento , L. , Ribeiro , A. , Tairum , C.A. , Jr. , Breyer , C.A. , Oliveira , M.A. , et al. . ; Therapeutic l-asparaginase: upstream, downstream and beyond ; Critical Reviews in Biotechnology , ( 2015 ); 37 ( 1 ): 1 – 18 . Google Scholar PubMed 7 Nath , C.E. , Dallapozza , L. , Eslick , A.E. , Misra , A. , Carr , D. , Earl , J.W. ; An isocratic fluorescence HPLC assay for the monitoring of l-asparaginase activity and l-asparagine depletion in children receiving E. coli l-asparaginase for the treatment of acute lymphoblastic leukaemia ; Biomedical Chromatography , ( 2009 ); 23 ( 2 ): 152 – 159 . Google Scholar Crossref Search ADS PubMed 8 Hawkins , D.S. , Park , J.R. , Thomson , B.G. , Felgenhauer , J.L. , Holcenberg , J.S. , Panosyan , E.H. , et al. . ; Asparaginase pharmacokinetics after intensive polyethylene glycol-conjugated l-asparaginase therapy for children with relapsed acute lymphoblastic leukemia ; Clinical Cancer Research , ( 2004 ); 10 ( 16 ): 5335 – 5341 . Google Scholar Crossref Search ADS PubMed 9 Panetta , J.C. , Gajjar , A. , Hijiya , N. , Hak , L.J. , Cheng , C. , Liu , W. , et al. . ; Comparison of native E. coli and PEG asparaginase pharmacokinetics and pharmacodynamics in pediatric acute lymphoblastic leukemia ; Clinical Pharmacology and Therapeutics , ( 2009 ); 86 ( 6 ): 651 – 658 . Google Scholar Crossref Search ADS PubMed 10 Avramis , V.I. , Sencer , S. , Periclou , A.P. , Sather , H. , Bostrom , B.C. , Cohen , L.J. , et al. . ; A randomized comparison of native Escherichia coli asparaginase and polyethylene glycol conjugated asparaginase for treatment of children with newly diagnosed standard-risk acute lymphoblastic leukemia: a Children’s Cancer Group study ; Blood , ( 2002 ); 99 ( 6 ): 1986 – 1994 . Google Scholar Crossref Search ADS PubMed 11 Asselin , B.L. , Whitin , J.C. , Coppola , D.J. , Rupp , I.P. , Sallan , S.E. , Cohen , H.J. ; Comparative pharmacokinetic studies of three asparaginase preparations ; Journal of Clinical Oncology , ( 1993 ); 11 ( 9 ): 1780 – 1786 . Google Scholar Crossref Search ADS PubMed 12 Avramis , V.I. , Panosyan , E.H. ; Pharmacokinetic/pharmacodynamic relationships of asparaginase formulations: the past, the present and recommendations for the future ; Clinical Pharmacokinetics , ( 2005 ); 44 ( 4 ): 367 – 393 . Google Scholar Crossref Search ADS PubMed 13 Pieters , R. , Appel , I. , Kuehnel , H.J. , Tetzlaff-Fohr , I. , Pichlmeier , U. , van der Vaart , I. , et al. . ; Pharmacokinetics, pharmacodynamics, efficacy, and safety of a new recombinant asparaginase preparation in children with previously untreated acute lymphoblastic leukemia: a randomized phase 2 clinical trial ; Blood , ( 2008 ); 112 ( 13 ): 4832 – 4838 . Google Scholar Crossref Search ADS PubMed 14 MacNeill , R. , Sangster , T. , Moussallie , M. , Trinh , V. , Stromeyer , R. , Daley , E. ; Stable-labeled analogues and reliable quantification of nonprotein biomarkers by LC-MS/MS ; Bioanalysis , ( 2010 ); 2 ( 1 ): 69 – 80 . Google Scholar Crossref Search ADS PubMed 15 Lanvers-Kaminsky , C. , Westhoff , P.S. , D'Incalci , M. , Zucchetti , M. , Boos , J. ; Immediate cooling does not prevent the ex vivo hydrolysis of l-asparagine by asparaginase ; Therapeutic Drug Monitoring , ( 2013 ); 36 ( 4 ): 549 – 552 . Google Scholar Crossref Search ADS 16 Liu , H. , Wang , F. , Xu , W. , May , K. , Richardson , D. ; Quantitation of asparagine deamidation by isotope labeling and liquid chromatography coupled with mass spectrometry analysis ; Analytical Biochemistry , ( 2013 ); 432 ( 1 ): 16 – 22 . Google Scholar Crossref Search ADS PubMed 17 Ferreira , A.A. , Ferraz , V. , Oliveira , P.M. , Godinho , A. , Silveira , D. , Raslan , D.S. ; Microwave-assisted derivatization and GC–MS analyses of amino acids from Ipomoea cairica aqueous extract ; Chemistry of Natural Compound , ( 2008 ); 44 ( 5 ): 679 – 681 . Google Scholar Crossref Search ADS 18 Ylikangas , P. , Mononen , I. ; A fluorometric assay for l-asparaginase activity and monitoring of l-asparaginase therapy ; Analytical Biochemistry , ( 2000 ); 280 ( 1 ): 42 – 45 . Google Scholar Crossref Search ADS PubMed 19 Yang , Z.H. , Wang , Q.Q. , Wang , X.Z. , Ou , L. , Song , H.F. ; Determination of l-asparagine in human serum on O-phthalaldehyde derivatives by RP-HPLC with pre-column derivatization ; Chinese Journal of Pharmaceutical Analysis , ( 2011 ); 31 ( 12 ): 2278 – 2283 . 20 Drabkova , P. , Andrlova , L. , Kand’ar , R. ; An HPLC method for the determination of selected amino acids in human embryo culture medium ; Biomedical Chromatography , ( 2017 ); 31 ( 2 ): 1 – 11 . Google Scholar Crossref Search ADS 21 Molnar-Perl , I. ; Advancement in the derivatizations of the amino groups with the O-phthaldehyde-thiol and with the 9-fluorenylmethyloxycarbonyl chloride reagents ; Journal of Chromatography B , ( 2011 ); 879 ( 17–18 ): 1241 – 1269 . Google Scholar Crossref Search ADS 22 Molnar-Perl , I. , Hanczko , R. , Koros , A. , Varga , Z. , Perl , A. ; Advances in the o-phthalaldehyde derivatization of amino acids and amines for their high performance liquid chromatographic analysis; 10th International Congress on Amino Acids and Proteins (ICAAP), Kallithea, Greece, August 20–25, (2007). 23 Molnár-Perl , I. , Vasanits , A. ; Stability and characteristics of the O-phthaldialdehyde/3-mercaptopropionic acid and O-phthaldialdehyde/N-acetyl-l-cysteine reagents and their amino acid derivatives measured by high-performance liquid chromatography ; Journal of Chromatography. A , ( 1999 ); 835 ( 1 ): 73 – 91 . Google Scholar Crossref Search ADS 24 Huang , G. , Hou , J. , Zhou , X. ; A measurement method for atmospheric ammonia and primary amines based on aqueous sampling, OPA derivatization and HPLC analysis ; Environmental Science & Technology , ( 2009 ); 43 ( 15 ): 5851 – 5856 . Google Scholar Crossref Search ADS PubMed 25 Stragierowicz , J. , Darago , A. , Brzeznicki , S. , Kilanowicz , A. ; Optimization of ultra-performance liquid choromatography (HPLC) with fluorescence detector (FLD) method for the quantitative determination of selected neurotransmitters in rat brain ; Medycyna Pracy , ( 2017 ); 68 ( 5 ): 583 – 591 . Google Scholar PubMed 26 Zandy , S.L. , Doherty , J.M. , Wibisono , N.D. , Gonzales , R.A. ; High sensitivity HPLC method for analysis of in vivo extracellular GABA using optimized fluorescence parameters for o-phthalaldehyde (OPA)/sulfite derivatives ; Journal of Chromatography B , ( 2017 ); 1055–1056 : 1 – 7 . Google Scholar Crossref Search ADS 27 Roth , M. ; Fluorescence reaction for amino acids ; Analytical Chemistry , ( 1971 ); 43 ( 7 ): 880 – 882 . Google Scholar Crossref Search ADS PubMed 28 Molnar-Perl , I. ; Derivatization and chromatographic behavior of the O-phthaldialdehyde amino acid derivatives obtained with various SH-group-containing additives ; Journal of Chromatography. A , ( 2001 ); 913 ( 1 ): 283 – 302 . Google Scholar Crossref Search ADS PubMed 29 Zacharis , C.K. , Theodoridis , G.A. , Voulgaropoulos , A.N. ; Coupling of sequential injection with liquid chromatography for the automated derivatization and on-line determination of amino acids ; Talanta , ( 2006 ); 69 ( 4 ): 841 – 847 . Google Scholar Crossref Search ADS PubMed 30 Jiang , H. , Long-Jun , G.U. , Chen , I. , Pan , C. , Xue , H.L. , Liang , A.B. , et al. . ; Contents of l-asparaginase-related amino acids in the plasma of children with ALL ; Chinese Journal of Contemporary Pediatrics , ( 2003 ); 5 ( 2 ): 81 – 85 . 31 U.S. Department of Health and Human Services Food and Drug Administration Guidance for Industry : Bioanalytical Method Validation http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm368107.pdf, 2013 (accessed July 7, 2017). 32 Tsurusawa , M. , Chin , M. , Iwai , A. , Nomura , K. , Maeba , H. , Taga , T. , et al. . ; l-Asparagine depletion levels and l-asparaginase activity in plasma of children with acute lymphoblastic leukemia under asparaginase treatment ; Cancer Chemotherapy and Pharmacology , ( 2004 ); 53 ( 3 ): 204 – 208 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. 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Optimization of a Precolumn OPA Derivatization HPLC Assay for Monitoring of l-Asparagine Depletion in Serum during l-Asparaginase Therapy

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Abstract

Abstract A method for monitoring l-asparagine (ASN) depletion in patients’ serum using reversed-phase high-performance liquid chromatography with precolumn o-phthalaldehyde and ethanethiol (ET) derivatization is described. In order to improve the signal and stability of analytes, several important factors including precipitant reagent, derivatization conditions and detection wavelengths were optimized. The recovery of the analytes in biological matrix was the highest when 4% sulfosalicylic acid (1:1, v/v) was used as a precipitant reagent. Optimal fluorescence detection parameters were determined as λex = 340 nm and λem = 444 nm for maximal signal. The signal of analytes was the highest when the reagent ET and borate buffer of pH 9.9 were used in the derivatization solution. And the corresponding derivative products were stable up to 19 h. The validated method had been successfully applied to monitor ASN depletion and l-aspartic acid, l-glutamine, l-glutamic acid levels in pediatric patients during l-asparaginase therapy. Introduction l-Asparaginase (ASNase) is one of the essential drugs in the treatment of childhood acute lymphoblastic leukemia (ALL) clinically (1–3). ASNase catalyzes the deamination of l-asparagine (ASN) and l-glutamine (GLN) to l-aspartic acid (ASP) and l-glutamic acid (GLU), respectively (4–7). The anticancer activity of ASNase is believed to be associated primarily with the depletion of ASN, but the second glutaminase activity has also been implicated in its anticancer mechanism of action and may enhance cell death (2, 4). Reports have shown that most of the patients could achieve satisfactory therapeutic effects when the concentration of ASN in serum was kept below 3 nmol/mL (8–10). However, ASNase therapy is associated with a range of adverse effects, including normal organs and tissues impaired by excessive depletion of ASN and GLN and hypersensitivity reactions (7, 9, 11, 12). The pharmacokinetic, pharmacodynamic and immunogenic properties of different preparations are characterized by wide intra- and inter-individual variabilities (13). Therefore, it is very important to implement the therapeutic drug monitoring during treatment with ASNase. There have been a number of assay methods for the determination of amino acids in biological samples, which include liquid chromatography coupled with mass spectrometry analysis (LC–MS) (14–16), gas chromatography–mass spectrometry analysis (GC–MS) (17) and high-performance liquid chromatography (HPLC) methods coupled with fluorescence detector (FLD) (7, 18, 19). Most amino acids neither contain any strong chromophores nor fluorophores in their molecule and therefore a suitable derivatization method is necessary (20). One of the most well-characterized derivatization reagents used for the analysis of amino acid is o-phthalaldehyde (OPA) (21, 22). The use of OPA conjunction with sulfhydryl reagent could furnish fast reaction with primary amines (R2NH2) in alkaline solution at ambient temperature to form sulfonatoisoindoles that have high selectivity and sensitivity with ultraviolet detector (UVD) or FLD, and the OPA/sulfhydryl reagent itself does not interfere with the detection of amino acids (21, 23, 24). OPA-HPLC-FLD assay is one of the widely used methods for the current determination of amino acids (20, 24–26). However, the instability of isoindoles produced from amino acids and OPA, in the presence of different sulfhydryl compounds, as well as the instability of the OPA–sulfhydryl reagent itself is known as two of the main disadvantages of OPA derivatization reaction (23, 27). Further, both the efficiency of derivatization and the stability of derivative products are affected by the sulfhydryl reagent type, the ratio of amino acid to derivatization reagent and the buffer pH (21, 23, 28, 29). Successful quantitative endogenous analytes without a true blank matrix is a daunting challenge currently with a limited number of possible solutions. Blank biological matrix was prepared by adding ASNase to deplete endogenous ASN in serum in some methods (14, 19). However, the matrix contained more endogenous ASP and cannot be used to analyze the correlation between ASN and ASP during treatment with ASNase. In other methods, the standard working solutions were directly used for method validation (7, 30). Consequently, the aim of the present study was to establish a sensitive HPLC method for quantification of ASP, ASN, GLU and GLN in patients’ serum, which could enable implementation of therapeutic drug monitoring during treatment with ASNase. In order to improve chromatographic separation and signals of the analytes, the gradient elution program, protein precipitation, sulfhydryl reagent, buffer pH, excitation wavelength and emission wavelength were optimized thoroughly. Additionally, the biological matrix was obtained by treating the healthy human serum with powdered activated carbon to reduce the levels of endogenous analytes. After OPA derivatization, ASP and ASN were measured with the internal standard method by FLD, while GLU and GLN were measured with the external standard method by UVD. Experimental section Chemicals and reagents ASN, ASP, GLN, GLU and carbocisteine (internal standard, IS) were obtained from Chinese food and Drug Inspection Institute (Beijing, China). 2-mercaptoethanol (MCE), 3-mercaptopropionic acid (MPA), ethanethiol (ET), and 5-sulfosalicylic acid and OPA were purchased from Sigma-Aldrich (Shanghai, China). HPLC-grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). Sodium acetate anhydrous, boric acid, sodium hydroxide and potassium chloride were of analytical grade and purchased from Nanjing Chemical Reagent Co. Ltd. (Nanjing, China). Ultrapure water was prepared using Milli-Q Advantage A10 System (Merck Millipore, MA, USA). Liquid chromatography Liquid chromatography was performed with a Shimadzu LC-10AD HPLC system consisting of an SIL-HTc autosampler, an LC-10ADvp delivery system and a CTO-10Avp column oven. The analytical column was ZORBAX Eclipse AAA column (150 × 4.6 mm, 5 μm) fitted with the precolumn (12.5 × 4.6 mm, 5 μm) of the same type. The mobile phase was composed of sodium acetate (0.05 M; pH 7.2) (A) and sodium acetate (0.1 M)–acetonitrile–methanol (46:44:10, v/v/v) (pH 7.2) (B) under gradient elution condition: 26% B at 0.00–3.00 min, 26–51% B at 3.00–15.00 min, 51% B at 15.00–16.00 min, 100% B at 16.01–20.00 min, and 26% B at 20.01–23 min. The flow rate was 1 mL/min, the injection volume was 5 μL and the temperature of column was maintained at 40°C. FLD and UVD detections Optimal excitation and emission wavelengths were determined for ASP and ASN by performing excitation and emission scan using the 10AXL FLD. GLU and GLN were detected by the SPD-M10Avp UVD at 338 nm. Preparation of solutions Standard stock solutions of ASN (2.0 mg/mL) and GLN (5.0 mg/mL) were prepared in 50% methanol including 1% HCl. Standard stock solutions of ASP (2.0 mg/mL), GLU (3.0 mg/mL) and the IS (1.0 mg/mL) were prepared in 0.2 M HCl. Those stock solutions were diluted with water to prepare standard working solutions. All solutions were stored at 4°C. The borate buffer solution was consisted of 0.8 M boric acid (dissolved in 0.8 M KCl solution) and 0.8 M NaOH (1:1, v/v; pH 9.9 ± 0.05). The derivatization reagent was prepared by mixing 1 mL of OPA solution (10 mg/mL, dissolved in methanol), 2 mL borate buffer and 54 μL ET (OPA–ET), or 16 μL MCE (OPA–MCE), or 19 μL MPA (OPA–MPA). It is important to note that OPA–ET reagent needs to be freshly prepared every 2 days, OPA–MCE and OPA–MPA reagents are stable within 9 days (21). Sample preparation Collected blood samples were immediately cooled in an ice-water bath to decant the serum. The 50 μL of serum was deproteinized immediately with 50 μL of 4% sulfosalicylic acid solution. After vortex for 30 s, the mixture was centrifuged at 16000 rpm at 4°C for 10 min. The supernatant was stored at −80°C until analysis. Prior to analysis, it was thawed at room temperature, and then the mixture was vortexed and centrifuged for 10 min at 16000 rpm at 4°C. A 5 μL aliquot of the IS (50 μg/mL) was added to 50 μL of the supernatant, and 10 μL of the mixed solution was diluted by 40 μL mobile phase and alkalized with 1 M NaOH (1 μL 1% phenolphthalein solution was used as the indicator). ASP, ASN, GLU and GLN were determined by RP-HPLC after precolumn derivatization with OPA–sulfhydryl reagent. The three different reagents (acetonitrile, 4% sulfosalicylic acid and 8% sulfosalicylic acid) used for protein precipitation were tested and compared with each other to find the best opportunity to guarantee a high recovery. QC samples were prepared by adding mixed standard solutions to treated biological matrix. The treated biological matrix was obtained by centrifugation after the healthy human serum was incubated with powdered activated carbon overnight under gentle agitation. Derivatization procedure The derivatization was performed by mixing 50 μL of diluted and alkalized standard or sample solution with the derivatization reagent. The mixed solution was vortexed 30 s and analyzed by HPLC system. Several important factors, including the variety of sulfhydryl reagents (MCE, MPA and ET), the volume ratio of amino acid to derivatization reagent (5:1, 5:3, 5:5 and 5:10) and the pH of buffer (8.5–11), were tested to achieve the best results. The stability of derivative products was tested by comparing the signal intensity at different time points between 0.5 min and 19 h after adding the derivatization reagent. Method valuation The method was validated according to the bioanalytical method validation guidance currently accepted by the US Food and Drug Administration (US FDA) (31). Calibration standards were prepared by spiking 5 μL of appropriate working mixture solutions with 45 μL of treated biological matrix. After OPA derivatization, ASP (1.41–135.2 nmol/mL) and ASN (0.76–90.83 nmol/mL) were quantified using the internal standard method, so the peak area ratio of the analyte to that of IS was determined in both treated biological matrix and treated biological matrix + standard. The difference of these two ratios was used to construct the calibration curve. GLU (8.50–815.6 nmol/mL) and GLN (12.83–1232 nmol/mL) were quantified using the external standard method, so the peak area of the analyte which had been corrected by corresponding value in treated biological matrix was used to construct the calibration curve. The recovery exercise was performed at all QC levels by comparing the response of processed QC samples with those of the processed treated biological matrix added the same amount of analytes and IS standard solutions. The precision and the accuracy of the method were evaluated at all QC levels and these samples were analyzed in three consecutive batches on different days. Then the intra- and the inter-batch precision were expressed as the relative standard deviation (%RSD) of each sample’s measured concentration, and the accuracy was defined as a percentage of the measured concentration over the theoretical value. Stability of ASP, ASN, GLU and GLN in standard solutions and deproteinizad serum extracts were analyzed in triplicate for three QC levels. The stability of stock solution of analytes and IS was evaluated at 4°C for 10 days. Further, the stability of all working solutions were tested at room temperature for 8 h. The short-time stability of deproteinized serum extracts of QC samples were evaluated at room temperature for 8 h. The post-preparative stability was evaluated by reanalyzing the derivatized samples placed in the autosampler at 22°C for 10 h. The freeze and thaw stability were evaluated by analyzing deproteinized serum extracts after they had been frozen (−80°C) and thawed (room temperature) on 3 consecutive days. The long-time stability of deproteinized serum extracts was evaluated at −80°C for 15 days. Clinical application The established method was used to monitor ASN depletion and the concentration change of ASP, GLU and GLN in the serum of pediatric patients with ALL who were receiving polyethylene glycol (PEG) ASNase treatment. The children with ALL were treatment with five-drug combination chemotherapy (dexamethasone + daunorubicin + vincristine + 6-mercaptopurine + PEG ASNase). PEG ASNase at 2000 U/m2 was injected intramuscularly. Blood samples were collected at different times, namely, before administration, within 7 days and 7 days after administration. This study had been approved by the ethics committee. All blood samples were obtained under the condition that the parents of all children who were selected for this study were previously informed to obtain consent and provided written informed consent. Results Optimization of excitation and emission wavelengths The excitation and emission spectra show that the optimal excitation and emission wavelengths for ASP and ASN derivatives were around 340 and 444 nm, respectively. In addition, the chromatogram revealed a very good signal response and baseline stability at 340/444 nm. Optimization of OPA derivatization conditions The experimental results indicated that the products derived from each target amino acid with the three sulfhydryl reagents (MCE, MPA and ET) were extremely different in chromatographic retention behavior and detector response. Among those derivatives, the retention time of OPA–MPA products were shortest, while OPA–MCE products were eluted in much longer time. OPA–ET products had highest signal response (Figure 1). Figure 1. View largeDownload slide Effects of different sulfhydryl reagents on amino acid derivative signal. Figure 1. View largeDownload slide Effects of different sulfhydryl reagents on amino acid derivative signal. The effect of borate buffer pH ranging from 8.5 to 11 on the derivatization reaction was studied. Finally, it was found that the borate buffer with the pH 9.9 was the most helpful basic medium to derivatization. The volume ratio of amino acid to derivatization reagent was also optimized. The signal of derivative products reached the highest when the volume ratio of amino acid solution to OPA–ET was 5:3. When the waiting time was increased to 19 h after adding the derivatization reagent, the detector responses of the target analytes were unchanged. The results from our experiment indicated that OPA–ET derivatives were relatively stable during the waiting time from 0.5 min to 19 h (Figure 2). Figure 2. View largeDownload slide Stability of OPA–ET/amino acid derivatives within 19 h. Figure 2. View largeDownload slide Stability of OPA–ET/amino acid derivatives within 19 h. Optimization of extraction conditions of the serum It was found that the sulfosalicylic acid provided a 17–37% higher yield for the four amino acids, comparing to the recovery obtained with acetonitrile. Then, sulfosalicylic acid extraction conditions were further optimized by changing its concentration and adding volume. As shown in Figure 3, the best recovery was obtained with 4% sulfosalicylic acid when it was added at 1:1 ratio to the QC sample for all the analytes except ASP. Figure 3. View largeDownload slide Effects of different volumes and concentrations of sulfosalicylic acid on amino acid extraction. Figure 3. View largeDownload slide Effects of different volumes and concentrations of sulfosalicylic acid on amino acid extraction. Method valuation Under the established HPLC–UVD–FLD conditions, no obvious endogenous interfering peaks at the elution times of ASP (5.9 min), ASN (13.2 min), GLU (7.9 min), GLN (14.4 min) and IS (9.9 min) were detected in real samples. Figure 4 presents representative chromatograms for ASN, ASP, GLN, GLU and IS resulting from the untreated biological matrix (Figure 4A), treated biological matrix (Figure 4B), QC sample (Figure 4C) and patient serum (Figure 4D). Figure 4. View largeDownload slide Representative chromatograms of OPA derivatives. ASN and ASP chromatograms obtained from the untreated biological matrix (A1), treated biological matrix (B1), QC sample (C1) and patient serum (D1). GLN and GLU chromatograms obtained from the untreated biological matrix (A2), treated biological matrix (B2), QC sample (C2) and patient serum (D2). Figure 4. View largeDownload slide Representative chromatograms of OPA derivatives. ASN and ASP chromatograms obtained from the untreated biological matrix (A1), treated biological matrix (B1), QC sample (C1) and patient serum (D1). GLN and GLU chromatograms obtained from the untreated biological matrix (A2), treated biological matrix (B2), QC sample (C2) and patient serum (D2). The extraction efficiency of three QC levels were consistent, precise and reproducible. The mean extraction recoveries ranged from 105% to 109% for ASP, 105% to 109% for ASN, 95% to 100% for GLU and 93% to 99% for GLN. In the assay of serum ASN, ASP, GLN and GLU, all the weighted calibration curves showed excellent linearity within tested concentration ranges, with the correlation coefficients (r) greater than 0.999. The equations of ASN, ASP, GLN and GLU calibration curve were C = 35.71 A/AIS + 0.24 (0.76–90.83 nmol/mL), C = 88.37 A/AIS + 0.29 (1.42–135.2 nmol/mL), C = 9.17 × 10–3 A − 1.63 (12.83–1232 nmol/mL) and C = 9.11 × 10–3 A − 0.73 (8.50–815.6 nmol/mL), respectively. The lower limit of quantification (LLOQ) of ASN was 0.76 nmol/mL which was measured from five independent samples and the precision (RSD = 9.34%) and accuracy (85.50–113.3%) were acceptable. The accuracy and the intra- and the inter-batch precision of the method were expressed as a percentage of the measured concentration over the theoretical value and a %RSD, respectively. The results indicated that the accuracy and the intra- and inter-batch precision were within the defined acceptance criteria (31). The accuracy ranged from 98.82% to 110.1%, intra- and inter-batch precision were less than 12% (Table I). Table I. The Precision and Accuracy of the Assay for Determining ASN, ASP, GLN, GLU in the Serum Concentration added (nmol/mL) Intra-day (n = 5) Inter-day (n = 15) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) ASP 135.2 138.5 ± 2.21 1.60 102.42 138.7 ± 2.41 1.74 102.55 18.03 19.04 ± 0.50 2.64 105.60 18.99 ± 0.40 2.10 105.32 2.82 2.96 ± 0.30 10.18 105.23 2.88 ± 0.28 9.73 102.16 ASN 90.83 90.74 ± 2.11 2.32 99.91 92.42 ± 2.03 2.20 101.75 12.11 12.23 ± 0.14 1.16 101.01 12.50 ± 0.28 2.28 103.20 1.89 1.88 ± 0.14 7.28 99.32 1.97 ± 0.17 8.76 103.98 GLU 815.6 824.8 ± 18.81 2.28 101.13 799.7 ± 24.75 3.10 98.05 108.7 114.3 ± 1.53 1.34 105.15 113.4 ± 5.18 4.57 104.24 16.99 16.95 ± 1.93 11.36 99.75 16.92 ± 1.83 10.82 99.55 GLN 1232 1217 ± 33.93 2.79 98.82 1198 ± 30.73 2.56 97.29 164.2 171.5 ± 4.02 2.35 104.41 164.5 ± 6.39 3.88 100.15 25.66 28.23 ± 1.45 5.15 110.03 26.72 ± 1.89 7.06 104.14 Concentration added (nmol/mL) Intra-day (n = 5) Inter-day (n = 15) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) ASP 135.2 138.5 ± 2.21 1.60 102.42 138.7 ± 2.41 1.74 102.55 18.03 19.04 ± 0.50 2.64 105.60 18.99 ± 0.40 2.10 105.32 2.82 2.96 ± 0.30 10.18 105.23 2.88 ± 0.28 9.73 102.16 ASN 90.83 90.74 ± 2.11 2.32 99.91 92.42 ± 2.03 2.20 101.75 12.11 12.23 ± 0.14 1.16 101.01 12.50 ± 0.28 2.28 103.20 1.89 1.88 ± 0.14 7.28 99.32 1.97 ± 0.17 8.76 103.98 GLU 815.6 824.8 ± 18.81 2.28 101.13 799.7 ± 24.75 3.10 98.05 108.7 114.3 ± 1.53 1.34 105.15 113.4 ± 5.18 4.57 104.24 16.99 16.95 ± 1.93 11.36 99.75 16.92 ± 1.83 10.82 99.55 GLN 1232 1217 ± 33.93 2.79 98.82 1198 ± 30.73 2.56 97.29 164.2 171.5 ± 4.02 2.35 104.41 164.5 ± 6.39 3.88 100.15 25.66 28.23 ± 1.45 5.15 110.03 26.72 ± 1.89 7.06 104.14 Table I. The Precision and Accuracy of the Assay for Determining ASN, ASP, GLN, GLU in the Serum Concentration added (nmol/mL) Intra-day (n = 5) Inter-day (n = 15) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) ASP 135.2 138.5 ± 2.21 1.60 102.42 138.7 ± 2.41 1.74 102.55 18.03 19.04 ± 0.50 2.64 105.60 18.99 ± 0.40 2.10 105.32 2.82 2.96 ± 0.30 10.18 105.23 2.88 ± 0.28 9.73 102.16 ASN 90.83 90.74 ± 2.11 2.32 99.91 92.42 ± 2.03 2.20 101.75 12.11 12.23 ± 0.14 1.16 101.01 12.50 ± 0.28 2.28 103.20 1.89 1.88 ± 0.14 7.28 99.32 1.97 ± 0.17 8.76 103.98 GLU 815.6 824.8 ± 18.81 2.28 101.13 799.7 ± 24.75 3.10 98.05 108.7 114.3 ± 1.53 1.34 105.15 113.4 ± 5.18 4.57 104.24 16.99 16.95 ± 1.93 11.36 99.75 16.92 ± 1.83 10.82 99.55 GLN 1232 1217 ± 33.93 2.79 98.82 1198 ± 30.73 2.56 97.29 164.2 171.5 ± 4.02 2.35 104.41 164.5 ± 6.39 3.88 100.15 25.66 28.23 ± 1.45 5.15 110.03 26.72 ± 1.89 7.06 104.14 Concentration added (nmol/mL) Intra-day (n = 5) Inter-day (n = 15) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) Concentration measured (mean ± SD nmol/mL) Precision (% RSD) Accuracy (%) ASP 135.2 138.5 ± 2.21 1.60 102.42 138.7 ± 2.41 1.74 102.55 18.03 19.04 ± 0.50 2.64 105.60 18.99 ± 0.40 2.10 105.32 2.82 2.96 ± 0.30 10.18 105.23 2.88 ± 0.28 9.73 102.16 ASN 90.83 90.74 ± 2.11 2.32 99.91 92.42 ± 2.03 2.20 101.75 12.11 12.23 ± 0.14 1.16 101.01 12.50 ± 0.28 2.28 103.20 1.89 1.88 ± 0.14 7.28 99.32 1.97 ± 0.17 8.76 103.98 GLU 815.6 824.8 ± 18.81 2.28 101.13 799.7 ± 24.75 3.10 98.05 108.7 114.3 ± 1.53 1.34 105.15 113.4 ± 5.18 4.57 104.24 16.99 16.95 ± 1.93 11.36 99.75 16.92 ± 1.83 10.82 99.55 GLN 1232 1217 ± 33.93 2.79 98.82 1198 ± 30.73 2.56 97.29 164.2 171.5 ± 4.02 2.35 104.41 164.5 ± 6.39 3.88 100.15 25.66 28.23 ± 1.45 5.15 110.03 26.72 ± 1.89 7.06 104.14 The results of stability tests at three QC levels (n = 3) are summarized in Table II. And the stability of the standard amino acid solutions are shown in Supplementary Table I. The data show reliable stability behavior of analytes and their derivative products under the condition tested. Table II. Stability of ASP, ASN, GLU, GLN in Human Serum at Different QC Levels (n = 3) Condition of sample analysis Concentration measured (mean ± SD) ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) 2.82 18.03 135.2 1.89 12.11 90.83 16.99 108.7 815.6 25.66 164.2 1232 Measured immediately (0 h) 3.22 ± 0.06 19.43 ± 0.16 138.0 ± 2.56 1.86 ± 0.15 12.43 ± 0.13 90.94 ± 2.09 15.31 ± 1.39 121.0 ± 2.78 773.3 ± 6.72 24.97 ± 4.02 163.8 ± 9.08 1261 ± 38.77 Short-term stability (8 h) 3.25 ± 0.03 19.33 ± 0.15 135.8 ± 1.64 1.91 ± 0.11 12.36 ± 0.14 89.03 ± 0.23 15.87 ± 1.37 123.1 ± 0.40 775.7 ± 3.00 28.96 ± 0.57 155.2 ± 1.87 1232 ± 26.79 Long-term stability (15 d) 3.00 ± 0.22 19.98 ± 0.47 146.1 ± 6.10 1.91 ± 0.17 12.85 ± 0.38 94.56 ± 1.99 15.26 ± 1.05 118.1 ± 3.70 765.4 ± 9.10 23.01 ± 2.35 176.5 ± 5.26 1258 ± 36.23 Post-preparative stability (10 h) 3.15 ± 0.10 17.92 ± 1.66 134.6 ± 5.67 1.76 ± 0.18 11.73 ± 0.80 88.60 ± 4.35 16.22 ± 1.85 111.1 ± 8.15 758.7 ± 15.16 23.84 ± 3.01 177.5 ± 5.72 1267 ± 32.69 Freeze and thaw stability (first time) 3.07 ± 0.22 18.98 ± 0.62 138.3 ± 2.12 1.90 ± 0.23 12.06 ± 0.47 87.55 ± 5.37 15.70 ± 1.36 120.0 ± 3.97 781.1 ± 13.31 22.40 ± 2.12 178.2 ± 6.37 1314 ± 30.84 Freeze and thaw stability (second time) 3.12 ± 0.13 18.98 ± 0.54 137.3 ± 3.27 2.00 ± 0.23 12.00 ± 0.50 87.71 ± 5.25 14.97 ± 0.86 119.1 ± 2.81 783.7 ± 18.74 22.15 ± 1.84 174.5 ± 4.57 1280 ± 63.94 Freeze and thaw stability (third time) 3.02 ± 0.19 18.53 ± 0.69 135.7 ± 3.44 2.03 ± 0.21 12.01 ± 0.50 87.69 ± 5.39 15.87 ± 1.29 118.7 ± 5.39 787.0 ± 15.49 22.96 ± 1.91 177.9 ± 5.20 1308 ± 53.91 Condition of sample analysis Concentration measured (mean ± SD) ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) 2.82 18.03 135.2 1.89 12.11 90.83 16.99 108.7 815.6 25.66 164.2 1232 Measured immediately (0 h) 3.22 ± 0.06 19.43 ± 0.16 138.0 ± 2.56 1.86 ± 0.15 12.43 ± 0.13 90.94 ± 2.09 15.31 ± 1.39 121.0 ± 2.78 773.3 ± 6.72 24.97 ± 4.02 163.8 ± 9.08 1261 ± 38.77 Short-term stability (8 h) 3.25 ± 0.03 19.33 ± 0.15 135.8 ± 1.64 1.91 ± 0.11 12.36 ± 0.14 89.03 ± 0.23 15.87 ± 1.37 123.1 ± 0.40 775.7 ± 3.00 28.96 ± 0.57 155.2 ± 1.87 1232 ± 26.79 Long-term stability (15 d) 3.00 ± 0.22 19.98 ± 0.47 146.1 ± 6.10 1.91 ± 0.17 12.85 ± 0.38 94.56 ± 1.99 15.26 ± 1.05 118.1 ± 3.70 765.4 ± 9.10 23.01 ± 2.35 176.5 ± 5.26 1258 ± 36.23 Post-preparative stability (10 h) 3.15 ± 0.10 17.92 ± 1.66 134.6 ± 5.67 1.76 ± 0.18 11.73 ± 0.80 88.60 ± 4.35 16.22 ± 1.85 111.1 ± 8.15 758.7 ± 15.16 23.84 ± 3.01 177.5 ± 5.72 1267 ± 32.69 Freeze and thaw stability (first time) 3.07 ± 0.22 18.98 ± 0.62 138.3 ± 2.12 1.90 ± 0.23 12.06 ± 0.47 87.55 ± 5.37 15.70 ± 1.36 120.0 ± 3.97 781.1 ± 13.31 22.40 ± 2.12 178.2 ± 6.37 1314 ± 30.84 Freeze and thaw stability (second time) 3.12 ± 0.13 18.98 ± 0.54 137.3 ± 3.27 2.00 ± 0.23 12.00 ± 0.50 87.71 ± 5.25 14.97 ± 0.86 119.1 ± 2.81 783.7 ± 18.74 22.15 ± 1.84 174.5 ± 4.57 1280 ± 63.94 Freeze and thaw stability (third time) 3.02 ± 0.19 18.53 ± 0.69 135.7 ± 3.44 2.03 ± 0.21 12.01 ± 0.50 87.69 ± 5.39 15.87 ± 1.29 118.7 ± 5.39 787.0 ± 15.49 22.96 ± 1.91 177.9 ± 5.20 1308 ± 53.91 Table II. Stability of ASP, ASN, GLU, GLN in Human Serum at Different QC Levels (n = 3) Condition of sample analysis Concentration measured (mean ± SD) ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) 2.82 18.03 135.2 1.89 12.11 90.83 16.99 108.7 815.6 25.66 164.2 1232 Measured immediately (0 h) 3.22 ± 0.06 19.43 ± 0.16 138.0 ± 2.56 1.86 ± 0.15 12.43 ± 0.13 90.94 ± 2.09 15.31 ± 1.39 121.0 ± 2.78 773.3 ± 6.72 24.97 ± 4.02 163.8 ± 9.08 1261 ± 38.77 Short-term stability (8 h) 3.25 ± 0.03 19.33 ± 0.15 135.8 ± 1.64 1.91 ± 0.11 12.36 ± 0.14 89.03 ± 0.23 15.87 ± 1.37 123.1 ± 0.40 775.7 ± 3.00 28.96 ± 0.57 155.2 ± 1.87 1232 ± 26.79 Long-term stability (15 d) 3.00 ± 0.22 19.98 ± 0.47 146.1 ± 6.10 1.91 ± 0.17 12.85 ± 0.38 94.56 ± 1.99 15.26 ± 1.05 118.1 ± 3.70 765.4 ± 9.10 23.01 ± 2.35 176.5 ± 5.26 1258 ± 36.23 Post-preparative stability (10 h) 3.15 ± 0.10 17.92 ± 1.66 134.6 ± 5.67 1.76 ± 0.18 11.73 ± 0.80 88.60 ± 4.35 16.22 ± 1.85 111.1 ± 8.15 758.7 ± 15.16 23.84 ± 3.01 177.5 ± 5.72 1267 ± 32.69 Freeze and thaw stability (first time) 3.07 ± 0.22 18.98 ± 0.62 138.3 ± 2.12 1.90 ± 0.23 12.06 ± 0.47 87.55 ± 5.37 15.70 ± 1.36 120.0 ± 3.97 781.1 ± 13.31 22.40 ± 2.12 178.2 ± 6.37 1314 ± 30.84 Freeze and thaw stability (second time) 3.12 ± 0.13 18.98 ± 0.54 137.3 ± 3.27 2.00 ± 0.23 12.00 ± 0.50 87.71 ± 5.25 14.97 ± 0.86 119.1 ± 2.81 783.7 ± 18.74 22.15 ± 1.84 174.5 ± 4.57 1280 ± 63.94 Freeze and thaw stability (third time) 3.02 ± 0.19 18.53 ± 0.69 135.7 ± 3.44 2.03 ± 0.21 12.01 ± 0.50 87.69 ± 5.39 15.87 ± 1.29 118.7 ± 5.39 787.0 ± 15.49 22.96 ± 1.91 177.9 ± 5.20 1308 ± 53.91 Condition of sample analysis Concentration measured (mean ± SD) ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) 2.82 18.03 135.2 1.89 12.11 90.83 16.99 108.7 815.6 25.66 164.2 1232 Measured immediately (0 h) 3.22 ± 0.06 19.43 ± 0.16 138.0 ± 2.56 1.86 ± 0.15 12.43 ± 0.13 90.94 ± 2.09 15.31 ± 1.39 121.0 ± 2.78 773.3 ± 6.72 24.97 ± 4.02 163.8 ± 9.08 1261 ± 38.77 Short-term stability (8 h) 3.25 ± 0.03 19.33 ± 0.15 135.8 ± 1.64 1.91 ± 0.11 12.36 ± 0.14 89.03 ± 0.23 15.87 ± 1.37 123.1 ± 0.40 775.7 ± 3.00 28.96 ± 0.57 155.2 ± 1.87 1232 ± 26.79 Long-term stability (15 d) 3.00 ± 0.22 19.98 ± 0.47 146.1 ± 6.10 1.91 ± 0.17 12.85 ± 0.38 94.56 ± 1.99 15.26 ± 1.05 118.1 ± 3.70 765.4 ± 9.10 23.01 ± 2.35 176.5 ± 5.26 1258 ± 36.23 Post-preparative stability (10 h) 3.15 ± 0.10 17.92 ± 1.66 134.6 ± 5.67 1.76 ± 0.18 11.73 ± 0.80 88.60 ± 4.35 16.22 ± 1.85 111.1 ± 8.15 758.7 ± 15.16 23.84 ± 3.01 177.5 ± 5.72 1267 ± 32.69 Freeze and thaw stability (first time) 3.07 ± 0.22 18.98 ± 0.62 138.3 ± 2.12 1.90 ± 0.23 12.06 ± 0.47 87.55 ± 5.37 15.70 ± 1.36 120.0 ± 3.97 781.1 ± 13.31 22.40 ± 2.12 178.2 ± 6.37 1314 ± 30.84 Freeze and thaw stability (second time) 3.12 ± 0.13 18.98 ± 0.54 137.3 ± 3.27 2.00 ± 0.23 12.00 ± 0.50 87.71 ± 5.25 14.97 ± 0.86 119.1 ± 2.81 783.7 ± 18.74 22.15 ± 1.84 174.5 ± 4.57 1280 ± 63.94 Freeze and thaw stability (third time) 3.02 ± 0.19 18.53 ± 0.69 135.7 ± 3.44 2.03 ± 0.21 12.01 ± 0.50 87.69 ± 5.39 15.87 ± 1.29 118.7 ± 5.39 787.0 ± 15.49 22.96 ± 1.91 177.9 ± 5.20 1308 ± 53.91 Clinical application Table III shows the level of ASN depletion and the change of concentration of ASP, GLU and GLN in pediatric patients before and after the course of therapy. Median ASN concentrations prior to treatment were 58.10 nmol/mL. After ASNase administration, ASN was consumed and its concentration was below the level of detection within 7 days, and since the 8th day, the ASN level began to rise and returned to the original state. In addition, after ASNase treatment, the concentration of GLN was significantly decreased, while the concentration of GLU was significantly increased, then the amino acids were also gradually returned to the original state from the 8th day. Table III. Monitoring ASN Depletion and the Concentration of ASP, GLU and GLN in ALL Children who Received 2000 U/m2 PEG Time of blood sampling ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) Median Median Median Median Range Range Range Range Prior treatment 62.01 58.10 154.1 367.3 (30.27–116.8) (29.14–141.3) (105.7–303.7) (187.7–455.2) (n = 9) (n = 9) (n = 9) (n = 9) Post-treatment (within 7 days) 64.62 N.D.a 384.7**b 136.4**b (23.96–86.19) (258.5–677.9) (22.23–204.2) (n = 12) (n = 12) (n = 12) (n = 12) Post-treatment (after 8 days) 46.02 69.01 163.3*c 301.2*c (33.95–67.85) (52.21–98.62) (49.44–174.7) (196.2–381.98) (n = 5) (n = 5) (n = 5) (n = 5) Time of blood sampling ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) Median Median Median Median Range Range Range Range Prior treatment 62.01 58.10 154.1 367.3 (30.27–116.8) (29.14–141.3) (105.7–303.7) (187.7–455.2) (n = 9) (n = 9) (n = 9) (n = 9) Post-treatment (within 7 days) 64.62 N.D.a 384.7**b 136.4**b (23.96–86.19) (258.5–677.9) (22.23–204.2) (n = 12) (n = 12) (n = 12) (n = 12) Post-treatment (after 8 days) 46.02 69.01 163.3*c 301.2*c (33.95–67.85) (52.21–98.62) (49.44–174.7) (196.2–381.98) (n = 5) (n = 5) (n = 5) (n = 5) aNot detection; *P < 0.01,**P < 0.001. bPrior treatment vs. post-treatment (within 7 days). cPost-treatment (within 7 days) vs. post-treatment (after 8 days). Table III. Monitoring ASN Depletion and the Concentration of ASP, GLU and GLN in ALL Children who Received 2000 U/m2 PEG Time of blood sampling ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) Median Median Median Median Range Range Range Range Prior treatment 62.01 58.10 154.1 367.3 (30.27–116.8) (29.14–141.3) (105.7–303.7) (187.7–455.2) (n = 9) (n = 9) (n = 9) (n = 9) Post-treatment (within 7 days) 64.62 N.D.a 384.7**b 136.4**b (23.96–86.19) (258.5–677.9) (22.23–204.2) (n = 12) (n = 12) (n = 12) (n = 12) Post-treatment (after 8 days) 46.02 69.01 163.3*c 301.2*c (33.95–67.85) (52.21–98.62) (49.44–174.7) (196.2–381.98) (n = 5) (n = 5) (n = 5) (n = 5) Time of blood sampling ASP (nmol/mL) ASN (nmol/mL) GLU (nmol/mL) GLN (nmol/mL) Median Median Median Median Range Range Range Range Prior treatment 62.01 58.10 154.1 367.3 (30.27–116.8) (29.14–141.3) (105.7–303.7) (187.7–455.2) (n = 9) (n = 9) (n = 9) (n = 9) Post-treatment (within 7 days) 64.62 N.D.a 384.7**b 136.4**b (23.96–86.19) (258.5–677.9) (22.23–204.2) (n = 12) (n = 12) (n = 12) (n = 12) Post-treatment (after 8 days) 46.02 69.01 163.3*c 301.2*c (33.95–67.85) (52.21–98.62) (49.44–174.7) (196.2–381.98) (n = 5) (n = 5) (n = 5) (n = 5) aNot detection; *P < 0.01,**P < 0.001. bPrior treatment vs. post-treatment (within 7 days). cPost-treatment (within 7 days) vs. post-treatment (after 8 days). Discussion Most of the analytical chemists, who had worked or are working in the field of amino acids and tried to use various derivatization techniques in their chromatographic determination, know that the OPA precolumn derivatization is a rapid and accurate method to determinate the concentrations of amino acids (21, 23, 28, 29). However, the instability of amino acid derivatives has attracted much attention (23, 29). In the experiment, three kinds of sulfhydryl reagents were compared. Among them, the OPA–ET derivatives exhibit the highest fluorescence and stability within 19 h. Petra Drábková et al. (20) found that the OPA–MCE derivatives are only stable for about half an hour and then a 30% decrease in fluorescence intensity was observed. Ibolya Molnár-Perl et al. (23) reported that OPA–MPA and OPA-N-acetyl-L-cysteine (NAC) derivatives are stable within 6 h. The optimal excitation and emission wavelengths were determined to guarantee the most sensitive fluorescence signal to detect the OPA–ET derivatives of ASN, ASP, GLN and GLU. The elution condition was optimized to achieve reliable peak separation in human serum samples, further confirming no interfering peaks were present in biological samples under the optimized elution program. We validated the method by quantifying analyte concentration using internal or external standards. The healthy human serum which incubated with activated carbon to reduce the endogenous analytes was used as a biological matrix to ensure the quantitative accuracy (Figure 4). The ASN and ASP concentrations could be quantified satisfactorily with FLD detection. Meanwhile, the concentration of GLN and GLU in the sample exceeded the range of the detector generating a flat-topped peak. Thus, using both UVD and FLD enables simultaneous quantification of all analytes. The limit of detection of ASN is 0.76 nmol/mL, which is qualified to monitor the depletion of serum ASN. The current method is optimized with the elution time of approximately 15 min, and another flush was utilized to wash out late eluting peaks. The run time is totally about 23 min, which is shorter than 35 min in the method reported by Christa E. Nath et al. (7). The effectiveness of the proposed method for monitoring ASN depletion during ASNase treatment was verified by analyzing the serum samples from the pediatric patient. Since the aim of ASNase therapy is to completely deplete the extracellular ASN, ASN concentration will always be under the threshold of 3 nmol/mL and close to the LOQ of the method (7, 9). It is worth mentioning that our method for ASN determination has good reproducibility (<10%) even at the low concentration of 1.89 nmol/mL. ASNase administration succeeded in depleting ASN concentrations to 0.76 nmol/mL or lower, and the change trend of four amino acid was in agreement with reference (7, 32). In summary, we have developed a relatively novel precolumn OPA derivatization HPLC assay which was qualified for the therapeutic drug monitoring during treatment with ASNase. Acknowledgments We would like to thank the patients who took part in this study and the all co-workers in Children’s Hospital of Nanjing Medical University and China Pharmaceutical University. Funding This project was financially supported by Medical Science and technology development Foundation, Nanjing Department of Health (No. YKK14112), the National Natural Science Foundation of China (No. 81403314) and the Natural Research Foundation of Jiangsu Province of china (No. BK20161456). Conflict of interest statement The authors declare no conflict of interest. References 1 Pui , C.H. , Evans , W.E. ; Treatment of acute lymphoblastic leukemia ; The New England Journal of Medicine , ( 2006 ); 354 ( 2 ): 166 – 178 . Google Scholar Crossref Search ADS PubMed 2 Purwaha , P. , Lorenzi , P.L. , Silva , L.P. , Hawke , D.H. , Weinstein , J.N. ; Targeted metabolomic analysis of amino acid response to l-asparaginase in adherent cells ; Metabolomics: Official Journal of the Metabolomic Society , ( 2014 ); 10 ( 5 ): 909 – 919 . Google Scholar PubMed 3 Muller , H.J. , Boos , J. ; Use of l-asparaginase in childhood ALL ; Critical Reviews in Oncology/Hematology , ( 1998 ); 28 ( 2 ): 97 – 113 . Google Scholar Crossref Search ADS PubMed 4 Miller , H.K. , Salser , J.S. , Balis , M.E. ; Amino acid levels following l-asparagine amidohydrolase (EC.3.5.1.1) Therapy ; Cancer Research , ( 1969 ); 29 ( 1 ): 183 – 187 . Google Scholar PubMed 5 Kawedia , J.D. , Rytting , M.E. ; Asparaginase in acute lymphoblastic leukemia ; Clinical Lymphoma, Myeloma & Leukemia , ( 2014 ); 14 ( S3 ): S14 – S17 . Google Scholar Crossref Search ADS PubMed 6 Lopes , A.M. , Oliveira-Nascimento , L. , Ribeiro , A. , Tairum , C.A. , Jr. , Breyer , C.A. , Oliveira , M.A. , et al. . ; Therapeutic l-asparaginase: upstream, downstream and beyond ; Critical Reviews in Biotechnology , ( 2015 ); 37 ( 1 ): 1 – 18 . Google Scholar PubMed 7 Nath , C.E. , Dallapozza , L. , Eslick , A.E. , Misra , A. , Carr , D. , Earl , J.W. ; An isocratic fluorescence HPLC assay for the monitoring of l-asparaginase activity and l-asparagine depletion in children receiving E. coli l-asparaginase for the treatment of acute lymphoblastic leukaemia ; Biomedical Chromatography , ( 2009 ); 23 ( 2 ): 152 – 159 . Google Scholar Crossref Search ADS PubMed 8 Hawkins , D.S. , Park , J.R. , Thomson , B.G. , Felgenhauer , J.L. , Holcenberg , J.S. , Panosyan , E.H. , et al. . ; Asparaginase pharmacokinetics after intensive polyethylene glycol-conjugated l-asparaginase therapy for children with relapsed acute lymphoblastic leukemia ; Clinical Cancer Research , ( 2004 ); 10 ( 16 ): 5335 – 5341 . Google Scholar Crossref Search ADS PubMed 9 Panetta , J.C. , Gajjar , A. , Hijiya , N. , Hak , L.J. , Cheng , C. , Liu , W. , et al. . ; Comparison of native E. coli and PEG asparaginase pharmacokinetics and pharmacodynamics in pediatric acute lymphoblastic leukemia ; Clinical Pharmacology and Therapeutics , ( 2009 ); 86 ( 6 ): 651 – 658 . Google Scholar Crossref Search ADS PubMed 10 Avramis , V.I. , Sencer , S. , Periclou , A.P. , Sather , H. , Bostrom , B.C. , Cohen , L.J. , et al. . ; A randomized comparison of native Escherichia coli asparaginase and polyethylene glycol conjugated asparaginase for treatment of children with newly diagnosed standard-risk acute lymphoblastic leukemia: a Children’s Cancer Group study ; Blood , ( 2002 ); 99 ( 6 ): 1986 – 1994 . Google Scholar Crossref Search ADS PubMed 11 Asselin , B.L. , Whitin , J.C. , Coppola , D.J. , Rupp , I.P. , Sallan , S.E. , Cohen , H.J. ; Comparative pharmacokinetic studies of three asparaginase preparations ; Journal of Clinical Oncology , ( 1993 ); 11 ( 9 ): 1780 – 1786 . Google Scholar Crossref Search ADS PubMed 12 Avramis , V.I. , Panosyan , E.H. ; Pharmacokinetic/pharmacodynamic relationships of asparaginase formulations: the past, the present and recommendations for the future ; Clinical Pharmacokinetics , ( 2005 ); 44 ( 4 ): 367 – 393 . Google Scholar Crossref Search ADS PubMed 13 Pieters , R. , Appel , I. , Kuehnel , H.J. , Tetzlaff-Fohr , I. , Pichlmeier , U. , van der Vaart , I. , et al. . ; Pharmacokinetics, pharmacodynamics, efficacy, and safety of a new recombinant asparaginase preparation in children with previously untreated acute lymphoblastic leukemia: a randomized phase 2 clinical trial ; Blood , ( 2008 ); 112 ( 13 ): 4832 – 4838 . Google Scholar Crossref Search ADS PubMed 14 MacNeill , R. , Sangster , T. , Moussallie , M. , Trinh , V. , Stromeyer , R. , Daley , E. ; Stable-labeled analogues and reliable quantification of nonprotein biomarkers by LC-MS/MS ; Bioanalysis , ( 2010 ); 2 ( 1 ): 69 – 80 . Google Scholar Crossref Search ADS PubMed 15 Lanvers-Kaminsky , C. , Westhoff , P.S. , D'Incalci , M. , Zucchetti , M. , Boos , J. ; Immediate cooling does not prevent the ex vivo hydrolysis of l-asparagine by asparaginase ; Therapeutic Drug Monitoring , ( 2013 ); 36 ( 4 ): 549 – 552 . Google Scholar Crossref Search ADS 16 Liu , H. , Wang , F. , Xu , W. , May , K. , Richardson , D. ; Quantitation of asparagine deamidation by isotope labeling and liquid chromatography coupled with mass spectrometry analysis ; Analytical Biochemistry , ( 2013 ); 432 ( 1 ): 16 – 22 . Google Scholar Crossref Search ADS PubMed 17 Ferreira , A.A. , Ferraz , V. , Oliveira , P.M. , Godinho , A. , Silveira , D. , Raslan , D.S. ; Microwave-assisted derivatization and GC–MS analyses of amino acids from Ipomoea cairica aqueous extract ; Chemistry of Natural Compound , ( 2008 ); 44 ( 5 ): 679 – 681 . Google Scholar Crossref Search ADS 18 Ylikangas , P. , Mononen , I. ; A fluorometric assay for l-asparaginase activity and monitoring of l-asparaginase therapy ; Analytical Biochemistry , ( 2000 ); 280 ( 1 ): 42 – 45 . Google Scholar Crossref Search ADS PubMed 19 Yang , Z.H. , Wang , Q.Q. , Wang , X.Z. , Ou , L. , Song , H.F. ; Determination of l-asparagine in human serum on O-phthalaldehyde derivatives by RP-HPLC with pre-column derivatization ; Chinese Journal of Pharmaceutical Analysis , ( 2011 ); 31 ( 12 ): 2278 – 2283 . 20 Drabkova , P. , Andrlova , L. , Kand’ar , R. ; An HPLC method for the determination of selected amino acids in human embryo culture medium ; Biomedical Chromatography , ( 2017 ); 31 ( 2 ): 1 – 11 . Google Scholar Crossref Search ADS 21 Molnar-Perl , I. ; Advancement in the derivatizations of the amino groups with the O-phthaldehyde-thiol and with the 9-fluorenylmethyloxycarbonyl chloride reagents ; Journal of Chromatography B , ( 2011 ); 879 ( 17–18 ): 1241 – 1269 . Google Scholar Crossref Search ADS 22 Molnar-Perl , I. , Hanczko , R. , Koros , A. , Varga , Z. , Perl , A. ; Advances in the o-phthalaldehyde derivatization of amino acids and amines for their high performance liquid chromatographic analysis; 10th International Congress on Amino Acids and Proteins (ICAAP), Kallithea, Greece, August 20–25, (2007). 23 Molnár-Perl , I. , Vasanits , A. ; Stability and characteristics of the O-phthaldialdehyde/3-mercaptopropionic acid and O-phthaldialdehyde/N-acetyl-l-cysteine reagents and their amino acid derivatives measured by high-performance liquid chromatography ; Journal of Chromatography. A , ( 1999 ); 835 ( 1 ): 73 – 91 . Google Scholar Crossref Search ADS 24 Huang , G. , Hou , J. , Zhou , X. ; A measurement method for atmospheric ammonia and primary amines based on aqueous sampling, OPA derivatization and HPLC analysis ; Environmental Science & Technology , ( 2009 ); 43 ( 15 ): 5851 – 5856 . Google Scholar Crossref Search ADS PubMed 25 Stragierowicz , J. , Darago , A. , Brzeznicki , S. , Kilanowicz , A. ; Optimization of ultra-performance liquid choromatography (HPLC) with fluorescence detector (FLD) method for the quantitative determination of selected neurotransmitters in rat brain ; Medycyna Pracy , ( 2017 ); 68 ( 5 ): 583 – 591 . Google Scholar PubMed 26 Zandy , S.L. , Doherty , J.M. , Wibisono , N.D. , Gonzales , R.A. ; High sensitivity HPLC method for analysis of in vivo extracellular GABA using optimized fluorescence parameters for o-phthalaldehyde (OPA)/sulfite derivatives ; Journal of Chromatography B , ( 2017 ); 1055–1056 : 1 – 7 . Google Scholar Crossref Search ADS 27 Roth , M. ; Fluorescence reaction for amino acids ; Analytical Chemistry , ( 1971 ); 43 ( 7 ): 880 – 882 . Google Scholar Crossref Search ADS PubMed 28 Molnar-Perl , I. ; Derivatization and chromatographic behavior of the O-phthaldialdehyde amino acid derivatives obtained with various SH-group-containing additives ; Journal of Chromatography. A , ( 2001 ); 913 ( 1 ): 283 – 302 . Google Scholar Crossref Search ADS PubMed 29 Zacharis , C.K. , Theodoridis , G.A. , Voulgaropoulos , A.N. ; Coupling of sequential injection with liquid chromatography for the automated derivatization and on-line determination of amino acids ; Talanta , ( 2006 ); 69 ( 4 ): 841 – 847 . Google Scholar Crossref Search ADS PubMed 30 Jiang , H. , Long-Jun , G.U. , Chen , I. , Pan , C. , Xue , H.L. , Liang , A.B. , et al. . ; Contents of l-asparaginase-related amino acids in the plasma of children with ALL ; Chinese Journal of Contemporary Pediatrics , ( 2003 ); 5 ( 2 ): 81 – 85 . 31 U.S. Department of Health and Human Services Food and Drug Administration Guidance for Industry : Bioanalytical Method Validation http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm368107.pdf, 2013 (accessed July 7, 2017). 32 Tsurusawa , M. , Chin , M. , Iwai , A. , Nomura , K. , Maeba , H. , Taga , T. , et al. . ; l-Asparagine depletion levels and l-asparaginase activity in plasma of children with acute lymphoblastic leukemia under asparaginase treatment ; Cancer Chemotherapy and Pharmacology , ( 2004 ); 53 ( 3 ): 204 – 208 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. 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