Determination of Acetaminophen, Dexchlorpheniramine, Caffeine, Cotinine and Salicylic acid in 100 μL of Whole Blood by UHPLC–MS/MS

Determination of Acetaminophen, Dexchlorpheniramine, Caffeine, Cotinine and Salicylic acid in 100... Abstract A sensitive and robust ultra-high-performance liquid chromatography–tandem mass spectrometry method has been developed and validated for the quantification of acetaminophen, dexchlorpheniramine, caffeine, cotinine and salicylic acid in postmortem blood samples from children younger than 4 years. The sample was prepared by a protein precipitation with ice-cold methanol/acetonitrile mixture (85:15, v/v). The organic phase was evaporated to dryness and the residue was dissolved in the mobile phase. Separation, with gradient elution and an acidic mobile phase, was achieved on an Acquity UPLC® HSS T3 column. The compounds were quantified using a multiple reaction-monitoring mode. Two transitions were monitored for each compound and one for the deuterated internal standards. The mass spectrometric detection in the positive ion mode was performed for all the compounds except salicylic acid which was detected in the negative ionization mode. The limits of quantification were as follows: acetaminophen 0.30 mg/L, dexchlorpheniramine 0.0050 mg/L, caffeine 0.099 mg/L, cotinine 0.00035 mg/L and salicylic acid 1.3 mg/L. Between-assay and within-assay precisions were ≤15% (biases: −10% to 26%) and ≤10%, respectively. Extraction recoveries varied from 93% to 137%. The matrix effects in blood, corrected with deuterated internal standards, were 100% ± 10% for all compounds except dexchlorpheniramine (111%) and caffeine (138%). Introduction The investigation of some drugs could be of greater importance in infants and children than in adults, as they are used more often in the pediatric population and because they possibly could be more toxic for infants and children. Over-the-counter as well as prescription cold and cough medications are often used in the pediatric population to ease infection symptoms of upper respiratory system. Data from the Norwegian Prescription Database indicate 52,000 prescriptions of respiratory system medications to children in the age of 0–4 years in 2015 (1). It has been reported that such medications were the predominant cause of death for infants (2, 3). Over-the-counter cough and cold medications were considered not be given to children younger than 2 years in a warning issued by the US Food and Drug Administration (4). Acetaminophen (paracetamol) is a common over-the-counter cold medication. It has been used for over 100 years as an antipyretic analgesic drug to children. Inappropriate dosing may lead to intoxication in children (5). Salicylic acid is another such analgesic used in the pediatric population. Dexchlorpheniramine belongs to a group of sedative antihistamines and is used in common cold and allergic treatments (6). Symptomatic treatment of lower respiratory tract infection with airways obstruction and cough using oral ephedrine was previously a widespread practice in Norway (7). Cotinine, the main metabolite of nicotine, is used as an indicator of exposure to side stream or active cigarette smoke. Ingestion of easily available smokeless tobacco products is a foremost reason for infant and child poisoning (8, 9). Parental smoking has been recognized as one of the most important environmental risk factors with regard to the sudden infant death syndrome (SIDS). Therefore, determination of cotinine is a valuable tool for determining tobacco exposure with possible implications for the manner of death (10–13). Caffeine is easily accessible through food, beverages and a number of medications. However, only few cases of acute caffeine poisoning in children population have been reported (14–16). These issues prompted us to develop a robust and sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for the quantitative determination of acetaminophen, dexchlorpheniramine, ephedrine, cotinine, caffeine and salicylic acid in 100 μL of whole blood from children autopsy cases. Experimental Chemicals and reagents Ammonium formate pro analysi was purchased from Merck KGaA (Darmstadt, Germany), formic acid 98%, GBR Rectapur from BDH Prolabo (Briare, France) and methanol, AR (analytical grade) from Lab-Scan (Poch SA, Gliwiche, Poland). Type 1 water (resistivity (MΩ-cm) at 25°C >18) was obtained from a Milli-Q UF Plus water purification system (Millipore, Bedford, MA, USA). Dexchlorpheniramine was purchased from Nycomed Pharma (Oslo, Norway) and Chiron AS (Trondheim, Norway), ephedrine from Fluka Chemicals (St. Louis, MO, USA) and Chiron AS, acetaminophen from NMD (Oslo, Norway) and Chiron AS, caffeine from RBI (MA, USA), salicylic acid from Sigma-Aldrich (St. Louise, MO, USA) and NMD and cotinine from Cerilliant (Round Rock, TX, USA) and Chiron AS. The internal standards cotinine-d3, acetaminophen-d4, ephedrine-d3, caffeine-d3 and salicylic acid-d4 were all purchased from Cerilliant. Chlorpheniramine-d6 was obtained from CDN Isotopes Inc. (Quebec, Canada). Calibrator and control sample (QC) solutions Stock solutions (1 mg/mL in methanol) were prepared for all the compounds and deuterated internal standards individually and stored at −20°C. Calibrators and controls were prepared from batches from different vendors, except caffeine, where only substance from one vendor was available at the time of method development. A mixture of the intermediate working solutions was prepared in methanol containing all analytes, except cotinine. Working solution of cotinine was made separately because of lower concentration level. Aqueous working solutions for calibrators (n = 5) and QC samples (n = 3) were prepared by dilution of the intermediate solutions with the mobile phase consisting of 10 mM ammonium formate buffer pH 3.1: methanol (90:10, v/v). A mixture of internal standards was prepared in the mobile phase (concentration range: 0.018–9.0 mg/L). All working solutions were stored at 4°C. The calibration range and the QC sample levels for each compound are shown in Table I. Table I. LOD, LOQ, calibration ranges, correlation coefficients (R2), within-assay precisions, between-assay precisions and biases Compound  LOD (mg/L)  LOQ (mg/L)  Calibration range (mg/L)  R2n = 10)  RSD (%)  QC sample concentration (mg/L)  Within-assay precision (%)  Between-assay precision (%)  Bias (%)  Cotinine  0.00021  0.00035  0.0010–0.10  0.9991  0.10  0.00070  1.6  5.1  14  0.0013  2.7  5.8  5.0  0.0035  1.3  3.7  2.1  Acetaminophen  0.19  0.30  1.5–15  0.9976  0.23  1.2  1.8  6.5  −3.4  2.3  1.3  6.4  −6.0  6.0  2.4  6.3  −8.2  Ephedrine  0.0089  0.015  0.060–0.60  0.9971  0.86  0.048  1.1  2.3  −1.5  0.090  1.5  3.7  −7.4  0.24  1.3  2.6  −10  Caffeine  0.062  0.099  0.39–3.9  0.9960  0.30  0.31  1.3  4.6  −0.2  0.58  1.3  7.4  −3.6  1.5  3.8  4.5  −2.4  Salicylic acid  0.86  1.3  1.4–14  0.9953  0.32  1.1  7.9  14  1.0  2.1  4.5  15  2.7  5.5  15  14  −6.4  Dexchlorpheniramine  0.0032  0.0050  0.020–0.20  0.9950  0.42  0.016  10  12  26  0.029  13  14  −6.7  0.078  8.1  12  0.3  Compound  LOD (mg/L)  LOQ (mg/L)  Calibration range (mg/L)  R2n = 10)  RSD (%)  QC sample concentration (mg/L)  Within-assay precision (%)  Between-assay precision (%)  Bias (%)  Cotinine  0.00021  0.00035  0.0010–0.10  0.9991  0.10  0.00070  1.6  5.1  14  0.0013  2.7  5.8  5.0  0.0035  1.3  3.7  2.1  Acetaminophen  0.19  0.30  1.5–15  0.9976  0.23  1.2  1.8  6.5  −3.4  2.3  1.3  6.4  −6.0  6.0  2.4  6.3  −8.2  Ephedrine  0.0089  0.015  0.060–0.60  0.9971  0.86  0.048  1.1  2.3  −1.5  0.090  1.5  3.7  −7.4  0.24  1.3  2.6  −10  Caffeine  0.062  0.099  0.39–3.9  0.9960  0.30  0.31  1.3  4.6  −0.2  0.58  1.3  7.4  −3.6  1.5  3.8  4.5  −2.4  Salicylic acid  0.86  1.3  1.4–14  0.9953  0.32  1.1  7.9  14  1.0  2.1  4.5  15  2.7  5.5  15  14  −6.4  Dexchlorpheniramine  0.0032  0.0050  0.020–0.20  0.9950  0.42  0.016  10  12  26  0.029  13  14  −6.7  0.078  8.1  12  0.3  Biological samples Sterilin® polystyrene tubes (20 mL) with polyethene screw caps (Bibby Sterilin, Staffordshire, UK) were used to collect blood samples from autopsies. A sample aliquot of 100 μL cardiac blood was transferred into 5 mL polypropylene tubes (Sarstedt AG, Rommelsdorf, Germany) and stored at 4°C until the time of analysis. Preparation of calibrators and control samples Calibrators, control and blank samples were chosen to be non-matrix based due to the difficulty of obtaining blank blood free from caffeine and cotinine. The calibrators, control- and blank samples were, therefore, prepared in a different manner than the blood samples. About 25 μL of internal standard working solution was added to 75 μL calibrator/QC working solutions in sample vials. For blank samples, 100 μL Type 1 water was used. The calibrator and control samples were well shaken to ensure no air bobbles present in the autosampler vials prior to injection. Preparation of biological samples About 25 μL of internal standard working solution was added to 100 μL whole blood samples. About 500 μL of ice-cold methanol/acetonitrile mixture (85:15, v/v) was added to each tube. Samples were then vortex-mixed briefly at high speed and kept in a freezer at −20°C for at least 10 min. The samples were centrifuged at 4°C for 10 min at 4500 rpm (3900 × g) and the organic layer was transferred to a glass tube. Zymark TurboVap (Caliper Life Sciences, Hopkinton, MA, USA) was used to evaporate the organic layer at 40°C with nitrogen at a pressure of 7 psi. About 100 μL of mobile phase ammonium formate buffer/methanol (90:10, v/v) was added to dissolve the residues prior to transferring to autosampler vials for analysis. The injection technique used was partial loop injections with a needle overfill flush with 2 μL injection volume. UHPLC–MS/MS conditions An Acquity UPLC system (Waters Corp., Milford, MA, USA) with a sample manager and a binary solvent manager was used. Chromatographic separation was carried out on an Acquity UPLC® HSS T3 column (2.1 mm ID × 50 mm, 1.7 μm particles) from Waters (Wexford, Ireland) operated at 65°C. The mobile phase consisted of 10 mM ammonium formate buffer, pH 3.1 (A) and methanol (B) with a flow rate 0.5 mL/min, Table II shows the gradient profile of the mobile phase. The assay was performed with a total cycle time of 6.3 min including the equilibration time. Multiple reaction-monitoring (MRM) transitions, cone voltages and collision energies for each compound were optimized by auto-calibration employing Intellistart software (Waters). Table II. MRM transitiona, retention time, cone voltage, collision energy, dwell time and gradient table Compound  MRM transition (m/z)  Retention time (min)  Cone voltage (V)  Collision energy (eV)  Dwell time (s)  Gradient table  Time (min)  A (%)  B (%)  Cotinine  177.1 > 80.1/98.1  1.31  35  20/20  0.022  0.00  90  10  Acetaminophen  152.1 > 110.0/92.8  1.45  30  16/24  0.022  0.50  90  10  Ephedrine  166.0 > 133.0/148.0  1.74  20  18/19  0.022  1.50  70  30  Caffeine  195.2 > 138.0/110.0  2.17  34  18/20  0.038  2.50  70  30  Salicylic acid  137.0 > 93.0/65.0  2.63  28  14/28  0.038  2.60  40  60  Dexchlorpheniramine  275.2 > 230.1/167.0  3.25  20  16/36  0.078  4.00  34  66              4.10  2  98              5.00  2  98              5.10  90  10  Compound  MRM transition (m/z)  Retention time (min)  Cone voltage (V)  Collision energy (eV)  Dwell time (s)  Gradient table  Time (min)  A (%)  B (%)  Cotinine  177.1 > 80.1/98.1  1.31  35  20/20  0.022  0.00  90  10  Acetaminophen  152.1 > 110.0/92.8  1.45  30  16/24  0.022  0.50  90  10  Ephedrine  166.0 > 133.0/148.0  1.74  20  18/19  0.022  1.50  70  30  Caffeine  195.2 > 138.0/110.0  2.17  34  18/20  0.038  2.50  70  30  Salicylic acid  137.0 > 93.0/65.0  2.63  28  14/28  0.038  2.60  40  60  Dexchlorpheniramine  275.2 > 230.1/167.0  3.25  20  16/36  0.078  4.00  34  66              4.10  2  98              5.00  2  98              5.10  90  10  aMRM transition used for quantification are in bold characters. A = 10 mM ammonium formate buffer, pH 3.1, B = MeOH. The MRM transitions were acquired in separate acquisition groups. Detection was performed on a Xevo TQ tandem mass spectrometer (Waters Corp., Milford, MA, USA) with electrospray ionization. The ion source block operated at 3 kV capillary voltage and 150°C. All compounds were detected in the positive ionization mode, except salicylic acid which was detected in negative ionization mode. Two MRM transitions monitored for each compound and one for the internal standards were used for identification and detection. Nitrogen was a desolvation gas obtained from a generator (OxymatN600, AGA, Norway, 99.93%) with a gas flow of 1100 L/h at 500°C. Argon was the collision gas delivered from the gas bottle (Argon, 99.999%, AGA, Norway) with pressure in collision cell held at ~3.5 × 10–3 mbar. Table II presents MRM transitions and associated mass spectrometric parameters (cone voltage, collision energy and dwell time) for the compounds and the internal standards. System operation and data acquisition were controlled by the MassLynx V4.1 software (Waters Corp., Milford, USA). TargetLynx quantification program (Waters Corp., Milford, USA) was used to process all data. Method validation Procedures for method validation were performed as proposed by Peters et al. (17) and Rivier (18) including linearity, within- and between-assay precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), matrix effects (MEs), extraction recovery (RE), selectivity of the method and carry-over. Calibration curves and quantification Quantification of the samples was done by integrating the peak height of the specific MRM chromatogram relative to the integrated peak height of respective internal standard. Five-point calibration curves of second order (y = ax2 + bx + c), with a weighing factor of 1/x and the origin excluded, were used. Calibration curve fits and standard deviations were determined as a mean of the correlation coefficient (R2) of 10 between-assay calibrations and presented in Table I. Precision and accuracy QC samples at three different concentration levels were analyzed for each compound to evaluate between-day precisions (n = 10, expressed as RSD (%)), accuracies (expressed as biases (%)) and within-assay precisions (repeatability, n = 10, expressed as RSD %). The results are shown in Table I. LOD and LOQ LOD and LOQ (n = 10) were determined by the analysis of dilutions of the lowest calibrator in 10 independent assays. The acceptance criteria for LOD and LOQ were the followings: the signal-to-noise ratio should be ≥3 and ≥10 for both MRM transitions, respectively. For LOD: the ion ratios, calculated as a ratio between the integrated peak height of the quantifier and qualifier MRM transitions, should be ±20% from the mean ion ratio of calibrators and QC samples. For LOQ: the quantitative value should be within ±15% from the nominal value. Selectivity Selectivity of the method was investigated by analyzing stock solutions of the compounds important in forensic analysis that could be present in samples (opiates, amphetamines, benzodiazepines, z-hypnotics, antidepressants, antipsychotics, antiepileptic drugs and designer drugs). Interferences and signal overlap were evaluated for each compound. The results are presented in Supplementary Table 1. MEs, RE and carry-over MEs and RE were investigated for all compounds at three concentration levels by the approach proposed by Matuszewski et al. (19). Three sets of samples were analyzed. Set 1 was a neat calibrator, Set 2 a post-extraction spiked blood and Set 3 a pre-extraction spiked blood. MEs were defined as ME% = peak heights from the samples spiked after the sample preparation (Set 2) divided by the peak heights from the set of neat calibrators (Set 1). A value below 100 indicates ion suppression and a value above 100 indicates ion enhancement. A total of five different lots of blank blood, previously determined to be negative for cotinine and caffeine, were used. In addition, the MEs were evaluated in peripheral and central blood, pericardial fluid, vastus lateralis muscle, psoas muscle and vitreous humor (n = 6) at one concentration level (approved by the Regional committee for research Ethics in Norway, reference number 2012/2073). RE (RE%) was calculated as a ratio between the integrated peak heights from the samples spiked before the sample preparation (Set 3) and the integrated peak heights from the samples spiked after the sample preparation (Set 2). An additional calibrator with a concentration two times higher than the highest calibrator was prepared for the examination of possible carry-over. Figure 1. View largeDownload slide Ion chromatograms for the blank sample and the lowest calibrator. Figure 1. View largeDownload slide Ion chromatograms for the blank sample and the lowest calibrator. Results and Discussion Sample preparation A rapid sample preparation was accomplished with a simple protein precipitation of 100 μL of blood sample with 0.5 mL ice-cold acetonitrile/methanol mixture (85:15 v/v). The organic layer was evaporated to dryness and dissolved in the mobile phase. The recoveries were in the range 98–110% for cotinine, 101–104% for acetaminophen, 90–100% for ephedrine, 116–137% for caffeine, 103–108% for salicylic acid and 93–99% for dexchlorpheniramine (Table III). Table III. MEs, RE in blood and MEs in different media Compound  MEs in blood (n = 5)  Corrected MEs in different autopsy materialsa (n = 6)  QC sample concentration (mg/L)  ME (%)  RSD (%)  Corrected ME (%)  Extraction recovery (%)  QC sample conc. (mg/L)  1  2  3  4  5  6  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  Cotinine  0.00088  100  4.8  99  110  0.0035  96  7.4  98  3.9  93  4.7  119  17  119  23  98  0.2  0.0035  87  3.0  98  98  0.11  88  2.8  98  104  Acetaminophen  1.5  103  4.2  102  104  6.0  101  3.0  100  2.2  99  2.6  101  1.1  101  2.5  100  1.8  6.0  96  1.7  99  101  15  93  3.2  101  101  Ephedrine  0.061  99  7.7  107  90  0.24  89  8.1  86  7.3  88  4.1  89  4.7  90  5.5  94  4.4  0.24  86  7.2  98  95  0.61  92  7.8  102  100  Caffeine  0.39  141  17  138  118  1.6  110  7.4  108  5.9  107  7.7  106  5.2  110  8.3  117  10  1.6  95  5.7  107  116  3.9  96  9.2  108  137  Salicylic acid  1.4  131  9.0  109  103  5.5  127  1.7  125  4.4  125  2.5  126  2.1  131  2.4  126  2.6  5.5  109  2.4  96  108  14  94  1.7  100  105  Dexchlorpheniramine  0.020  81  3.8  101  99  0.078  101  2.1  99  5.1  96  4.4  91  7.0  93  12.1  103  2.4  0.078  75  3.0  103  94  0.20  84  3.2  111  93  Compound  MEs in blood (n = 5)  Corrected MEs in different autopsy materialsa (n = 6)  QC sample concentration (mg/L)  ME (%)  RSD (%)  Corrected ME (%)  Extraction recovery (%)  QC sample conc. (mg/L)  1  2  3  4  5  6  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  Cotinine  0.00088  100  4.8  99  110  0.0035  96  7.4  98  3.9  93  4.7  119  17  119  23  98  0.2  0.0035  87  3.0  98  98  0.11  88  2.8  98  104  Acetaminophen  1.5  103  4.2  102  104  6.0  101  3.0  100  2.2  99  2.6  101  1.1  101  2.5  100  1.8  6.0  96  1.7  99  101  15  93  3.2  101  101  Ephedrine  0.061  99  7.7  107  90  0.24  89  8.1  86  7.3  88  4.1  89  4.7  90  5.5  94  4.4  0.24  86  7.2  98  95  0.61  92  7.8  102  100  Caffeine  0.39  141  17  138  118  1.6  110  7.4  108  5.9  107  7.7  106  5.2  110  8.3  117  10  1.6  95  5.7  107  116  3.9  96  9.2  108  137  Salicylic acid  1.4  131  9.0  109  103  5.5  127  1.7  125  4.4  125  2.5  126  2.1  131  2.4  126  2.6  5.5  109  2.4  96  108  14  94  1.7  100  105  Dexchlorpheniramine  0.020  81  3.8  101  99  0.078  101  2.1  99  5.1  96  4.4  91  7.0  93  12.1  103  2.4  0.078  75  3.0  103  94  0.20  84  3.2  111  93  aDifferent media: 1 = peripheral blood, 2 = central blood, 3 = pericardial fluid, 4 = vastus lateralis muscle, 5 = psoas muscle and 6 = vitreous humor. Quantification and calibration curves A choice of preparing the blank samples, calibrators and the QC samples in mobile phase instead of in blood was made at the early stage of method development (Figure 1). Although the use of matrix-matched calibrators and samples is the ideal approach, the possibility to use water calibrators instead of whole blood calibrators has previously been demonstrated for several drugs of abuse (20). The use of dedicated isotope-labeled internal standards will, in addition, help to compensate for differences between the calibrators and samples. For salicylic acid and acetaminophen, proficiency testing has demonstrated the suitability of the method for analysis of whole blood. The fact that cotinine and specially caffeine were present in a large number of tested blank blood batches made this choice necessary to facilitate routine application of the method. The calibration curves (n = 10), created from five concentration levels, were found to be reproducible with RSDs ≤0.9% for all compounds in the given concentration ranges. Correlation coefficients were ≥0.995 for all the compounds. Calibration ranges, correlation coefficients R2 and RSDs of correlation coefficients are shown in Table I. Precision, accuracy, LOD and LOQ The within-assay precisions were between 1.1% and 15% and the between-assay precisions between 2.3% and 15%. The biases for the QC samples were within ±10% for all compounds, except for cotinine (14%) and dexchlorpheniramine (26%) at the lowest QC levels. The calculated precisions and biases together with LOD and LOQ values were found satisfactory (Table I). MEs, extraction recoveries and carry-over The residual matrix components still present after sample preparation have an influence on the MS ionization processes and may increase (ion enhancement) or decrease (ion suppression) the analytical signal (21). Five different blank blood batches and six different autopsy matrixes were used to determine the MEs. The results are presented in Table III. Deuterated internal standards were used for calculating the corrected MEs in blood. For all the compounds, the MEs were 100 ± 10% except caffeine (138%) and dexchlorpheniramine (111%). Slight ion suppression was observed for ephedrine in peripheral and central blood, pericardial fluid and vastus lateralis muscle from the autopsy cases. Cotinine showed ion enhancement in vastus lateralis and psoas muscle, and salicylic acid in all six autopsy matrixes. Extraction recoveries were found to be above 90% for all the compounds (Table III). Carry-over was investigated by analyzing a blank sample after a sample spiked to times higher than the highest calibrator. Carry-over was not observed for any of the compounds. Stability Stability experiments including freeze/thaw stability and long-term stability have not been performed. Several stability studies are available from the literature for cotinine (22–24), chlorpheniramine (dexchlorpheniramine is a dextrorotatory isomer of chlorpheniramine) (25), ephedrine (26–28), acetaminophen (29) and salicylic acid (30), indicating a good stability. Application of the method We have analyzed 119 samples from infants and children (≤4 years) during the period 2012–2016. Sixty-three cases (47%) were negative. Caffeine was detected solely in 15 (27%), cotinine in 32 (57%) and acetaminophen in 21 (38%) cases. Acetaminophen together with caffeine was found in two cases and together with dexchlorpheniramine in one case. Both acetaminophen and cotinine were present in two cases and caffeine and cotinine in five cases. Caffeine, cotinine and acetaminophen were proven in one case. Ephedrine and salicylic acid were not found in any of the samples. The data above are depersonalized data, so we do not know if there were other drugs present in these samples. Neither do we know the cause of deaths (i.e., SIDS, sudden unexpected death in childhood, accident). The samples received were varying: peripheral blood, central blood, thorax blood or fluid, blood from abdomen, blood from skull base, pericardial fluid, pleural effusion fluid and blood from hospital admission. Quantified concentrations are, therefore, not reported. Conclusions A fast, selective, and robust UHPLC–MS/MS method for the determination of acetaminophen, dexchlorpheniramine, caffeine, cotinine and salicylic acid in 100 μL of whole blood has been developed. The method was validated with the calibrator, control and blank samples prepared in the mobile phase instead of blood matrix because of caffeine and cotinine-free human blank blood is difficult to obtain. The deuterated internal standards coeluted with the compounds of interest, corrected MEs and compensated possible loses during the preparation of biological samples. The method performs well in routine use with simple procedure for sample preparation, good sensitivity and short total cycle time. We have not found any previously published method from the literature including this combination of compounds. Supplementary data Supplementary data are available at Journal of Analytical Toxicology online. Acknowledgments We would like to thank Lena Kristoffersen and Åse-Marit Leere Øiestad for advice and critical reading of the article. References 1 The Norwegian Prescription Database. http://www.norpd.no/ (accessed Feb 28, 2017). 2 Srinivasan, A., Budnitz, D., Shehab, N., Cohen, A. ( 2007) Infant deaths associated with cough and cold medications—two states, 2005 (Reprinted from MMWR, 56, 1–4, 2007). Journal of the American Medical Directors Association , 297, 800– 801. Google Scholar CrossRef Search ADS   3 Rimsza, M.E., Newberry, S. ( 2008) Unexpected infant deaths associated with use of cough and cold medications. Pediatrics , 122, E318– E322. 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For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Analytical Toxicology Oxford University Press

Determination of Acetaminophen, Dexchlorpheniramine, Caffeine, Cotinine and Salicylic acid in 100 μL of Whole Blood by UHPLC–MS/MS

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Oxford University Press
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© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0146-4760
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1945-2403
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10.1093/jat/bkx089
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Abstract

Abstract A sensitive and robust ultra-high-performance liquid chromatography–tandem mass spectrometry method has been developed and validated for the quantification of acetaminophen, dexchlorpheniramine, caffeine, cotinine and salicylic acid in postmortem blood samples from children younger than 4 years. The sample was prepared by a protein precipitation with ice-cold methanol/acetonitrile mixture (85:15, v/v). The organic phase was evaporated to dryness and the residue was dissolved in the mobile phase. Separation, with gradient elution and an acidic mobile phase, was achieved on an Acquity UPLC® HSS T3 column. The compounds were quantified using a multiple reaction-monitoring mode. Two transitions were monitored for each compound and one for the deuterated internal standards. The mass spectrometric detection in the positive ion mode was performed for all the compounds except salicylic acid which was detected in the negative ionization mode. The limits of quantification were as follows: acetaminophen 0.30 mg/L, dexchlorpheniramine 0.0050 mg/L, caffeine 0.099 mg/L, cotinine 0.00035 mg/L and salicylic acid 1.3 mg/L. Between-assay and within-assay precisions were ≤15% (biases: −10% to 26%) and ≤10%, respectively. Extraction recoveries varied from 93% to 137%. The matrix effects in blood, corrected with deuterated internal standards, were 100% ± 10% for all compounds except dexchlorpheniramine (111%) and caffeine (138%). Introduction The investigation of some drugs could be of greater importance in infants and children than in adults, as they are used more often in the pediatric population and because they possibly could be more toxic for infants and children. Over-the-counter as well as prescription cold and cough medications are often used in the pediatric population to ease infection symptoms of upper respiratory system. Data from the Norwegian Prescription Database indicate 52,000 prescriptions of respiratory system medications to children in the age of 0–4 years in 2015 (1). It has been reported that such medications were the predominant cause of death for infants (2, 3). Over-the-counter cough and cold medications were considered not be given to children younger than 2 years in a warning issued by the US Food and Drug Administration (4). Acetaminophen (paracetamol) is a common over-the-counter cold medication. It has been used for over 100 years as an antipyretic analgesic drug to children. Inappropriate dosing may lead to intoxication in children (5). Salicylic acid is another such analgesic used in the pediatric population. Dexchlorpheniramine belongs to a group of sedative antihistamines and is used in common cold and allergic treatments (6). Symptomatic treatment of lower respiratory tract infection with airways obstruction and cough using oral ephedrine was previously a widespread practice in Norway (7). Cotinine, the main metabolite of nicotine, is used as an indicator of exposure to side stream or active cigarette smoke. Ingestion of easily available smokeless tobacco products is a foremost reason for infant and child poisoning (8, 9). Parental smoking has been recognized as one of the most important environmental risk factors with regard to the sudden infant death syndrome (SIDS). Therefore, determination of cotinine is a valuable tool for determining tobacco exposure with possible implications for the manner of death (10–13). Caffeine is easily accessible through food, beverages and a number of medications. However, only few cases of acute caffeine poisoning in children population have been reported (14–16). These issues prompted us to develop a robust and sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for the quantitative determination of acetaminophen, dexchlorpheniramine, ephedrine, cotinine, caffeine and salicylic acid in 100 μL of whole blood from children autopsy cases. Experimental Chemicals and reagents Ammonium formate pro analysi was purchased from Merck KGaA (Darmstadt, Germany), formic acid 98%, GBR Rectapur from BDH Prolabo (Briare, France) and methanol, AR (analytical grade) from Lab-Scan (Poch SA, Gliwiche, Poland). Type 1 water (resistivity (MΩ-cm) at 25°C >18) was obtained from a Milli-Q UF Plus water purification system (Millipore, Bedford, MA, USA). Dexchlorpheniramine was purchased from Nycomed Pharma (Oslo, Norway) and Chiron AS (Trondheim, Norway), ephedrine from Fluka Chemicals (St. Louis, MO, USA) and Chiron AS, acetaminophen from NMD (Oslo, Norway) and Chiron AS, caffeine from RBI (MA, USA), salicylic acid from Sigma-Aldrich (St. Louise, MO, USA) and NMD and cotinine from Cerilliant (Round Rock, TX, USA) and Chiron AS. The internal standards cotinine-d3, acetaminophen-d4, ephedrine-d3, caffeine-d3 and salicylic acid-d4 were all purchased from Cerilliant. Chlorpheniramine-d6 was obtained from CDN Isotopes Inc. (Quebec, Canada). Calibrator and control sample (QC) solutions Stock solutions (1 mg/mL in methanol) were prepared for all the compounds and deuterated internal standards individually and stored at −20°C. Calibrators and controls were prepared from batches from different vendors, except caffeine, where only substance from one vendor was available at the time of method development. A mixture of the intermediate working solutions was prepared in methanol containing all analytes, except cotinine. Working solution of cotinine was made separately because of lower concentration level. Aqueous working solutions for calibrators (n = 5) and QC samples (n = 3) were prepared by dilution of the intermediate solutions with the mobile phase consisting of 10 mM ammonium formate buffer pH 3.1: methanol (90:10, v/v). A mixture of internal standards was prepared in the mobile phase (concentration range: 0.018–9.0 mg/L). All working solutions were stored at 4°C. The calibration range and the QC sample levels for each compound are shown in Table I. Table I. LOD, LOQ, calibration ranges, correlation coefficients (R2), within-assay precisions, between-assay precisions and biases Compound  LOD (mg/L)  LOQ (mg/L)  Calibration range (mg/L)  R2n = 10)  RSD (%)  QC sample concentration (mg/L)  Within-assay precision (%)  Between-assay precision (%)  Bias (%)  Cotinine  0.00021  0.00035  0.0010–0.10  0.9991  0.10  0.00070  1.6  5.1  14  0.0013  2.7  5.8  5.0  0.0035  1.3  3.7  2.1  Acetaminophen  0.19  0.30  1.5–15  0.9976  0.23  1.2  1.8  6.5  −3.4  2.3  1.3  6.4  −6.0  6.0  2.4  6.3  −8.2  Ephedrine  0.0089  0.015  0.060–0.60  0.9971  0.86  0.048  1.1  2.3  −1.5  0.090  1.5  3.7  −7.4  0.24  1.3  2.6  −10  Caffeine  0.062  0.099  0.39–3.9  0.9960  0.30  0.31  1.3  4.6  −0.2  0.58  1.3  7.4  −3.6  1.5  3.8  4.5  −2.4  Salicylic acid  0.86  1.3  1.4–14  0.9953  0.32  1.1  7.9  14  1.0  2.1  4.5  15  2.7  5.5  15  14  −6.4  Dexchlorpheniramine  0.0032  0.0050  0.020–0.20  0.9950  0.42  0.016  10  12  26  0.029  13  14  −6.7  0.078  8.1  12  0.3  Compound  LOD (mg/L)  LOQ (mg/L)  Calibration range (mg/L)  R2n = 10)  RSD (%)  QC sample concentration (mg/L)  Within-assay precision (%)  Between-assay precision (%)  Bias (%)  Cotinine  0.00021  0.00035  0.0010–0.10  0.9991  0.10  0.00070  1.6  5.1  14  0.0013  2.7  5.8  5.0  0.0035  1.3  3.7  2.1  Acetaminophen  0.19  0.30  1.5–15  0.9976  0.23  1.2  1.8  6.5  −3.4  2.3  1.3  6.4  −6.0  6.0  2.4  6.3  −8.2  Ephedrine  0.0089  0.015  0.060–0.60  0.9971  0.86  0.048  1.1  2.3  −1.5  0.090  1.5  3.7  −7.4  0.24  1.3  2.6  −10  Caffeine  0.062  0.099  0.39–3.9  0.9960  0.30  0.31  1.3  4.6  −0.2  0.58  1.3  7.4  −3.6  1.5  3.8  4.5  −2.4  Salicylic acid  0.86  1.3  1.4–14  0.9953  0.32  1.1  7.9  14  1.0  2.1  4.5  15  2.7  5.5  15  14  −6.4  Dexchlorpheniramine  0.0032  0.0050  0.020–0.20  0.9950  0.42  0.016  10  12  26  0.029  13  14  −6.7  0.078  8.1  12  0.3  Biological samples Sterilin® polystyrene tubes (20 mL) with polyethene screw caps (Bibby Sterilin, Staffordshire, UK) were used to collect blood samples from autopsies. A sample aliquot of 100 μL cardiac blood was transferred into 5 mL polypropylene tubes (Sarstedt AG, Rommelsdorf, Germany) and stored at 4°C until the time of analysis. Preparation of calibrators and control samples Calibrators, control and blank samples were chosen to be non-matrix based due to the difficulty of obtaining blank blood free from caffeine and cotinine. The calibrators, control- and blank samples were, therefore, prepared in a different manner than the blood samples. About 25 μL of internal standard working solution was added to 75 μL calibrator/QC working solutions in sample vials. For blank samples, 100 μL Type 1 water was used. The calibrator and control samples were well shaken to ensure no air bobbles present in the autosampler vials prior to injection. Preparation of biological samples About 25 μL of internal standard working solution was added to 100 μL whole blood samples. About 500 μL of ice-cold methanol/acetonitrile mixture (85:15, v/v) was added to each tube. Samples were then vortex-mixed briefly at high speed and kept in a freezer at −20°C for at least 10 min. The samples were centrifuged at 4°C for 10 min at 4500 rpm (3900 × g) and the organic layer was transferred to a glass tube. Zymark TurboVap (Caliper Life Sciences, Hopkinton, MA, USA) was used to evaporate the organic layer at 40°C with nitrogen at a pressure of 7 psi. About 100 μL of mobile phase ammonium formate buffer/methanol (90:10, v/v) was added to dissolve the residues prior to transferring to autosampler vials for analysis. The injection technique used was partial loop injections with a needle overfill flush with 2 μL injection volume. UHPLC–MS/MS conditions An Acquity UPLC system (Waters Corp., Milford, MA, USA) with a sample manager and a binary solvent manager was used. Chromatographic separation was carried out on an Acquity UPLC® HSS T3 column (2.1 mm ID × 50 mm, 1.7 μm particles) from Waters (Wexford, Ireland) operated at 65°C. The mobile phase consisted of 10 mM ammonium formate buffer, pH 3.1 (A) and methanol (B) with a flow rate 0.5 mL/min, Table II shows the gradient profile of the mobile phase. The assay was performed with a total cycle time of 6.3 min including the equilibration time. Multiple reaction-monitoring (MRM) transitions, cone voltages and collision energies for each compound were optimized by auto-calibration employing Intellistart software (Waters). Table II. MRM transitiona, retention time, cone voltage, collision energy, dwell time and gradient table Compound  MRM transition (m/z)  Retention time (min)  Cone voltage (V)  Collision energy (eV)  Dwell time (s)  Gradient table  Time (min)  A (%)  B (%)  Cotinine  177.1 > 80.1/98.1  1.31  35  20/20  0.022  0.00  90  10  Acetaminophen  152.1 > 110.0/92.8  1.45  30  16/24  0.022  0.50  90  10  Ephedrine  166.0 > 133.0/148.0  1.74  20  18/19  0.022  1.50  70  30  Caffeine  195.2 > 138.0/110.0  2.17  34  18/20  0.038  2.50  70  30  Salicylic acid  137.0 > 93.0/65.0  2.63  28  14/28  0.038  2.60  40  60  Dexchlorpheniramine  275.2 > 230.1/167.0  3.25  20  16/36  0.078  4.00  34  66              4.10  2  98              5.00  2  98              5.10  90  10  Compound  MRM transition (m/z)  Retention time (min)  Cone voltage (V)  Collision energy (eV)  Dwell time (s)  Gradient table  Time (min)  A (%)  B (%)  Cotinine  177.1 > 80.1/98.1  1.31  35  20/20  0.022  0.00  90  10  Acetaminophen  152.1 > 110.0/92.8  1.45  30  16/24  0.022  0.50  90  10  Ephedrine  166.0 > 133.0/148.0  1.74  20  18/19  0.022  1.50  70  30  Caffeine  195.2 > 138.0/110.0  2.17  34  18/20  0.038  2.50  70  30  Salicylic acid  137.0 > 93.0/65.0  2.63  28  14/28  0.038  2.60  40  60  Dexchlorpheniramine  275.2 > 230.1/167.0  3.25  20  16/36  0.078  4.00  34  66              4.10  2  98              5.00  2  98              5.10  90  10  aMRM transition used for quantification are in bold characters. A = 10 mM ammonium formate buffer, pH 3.1, B = MeOH. The MRM transitions were acquired in separate acquisition groups. Detection was performed on a Xevo TQ tandem mass spectrometer (Waters Corp., Milford, MA, USA) with electrospray ionization. The ion source block operated at 3 kV capillary voltage and 150°C. All compounds were detected in the positive ionization mode, except salicylic acid which was detected in negative ionization mode. Two MRM transitions monitored for each compound and one for the internal standards were used for identification and detection. Nitrogen was a desolvation gas obtained from a generator (OxymatN600, AGA, Norway, 99.93%) with a gas flow of 1100 L/h at 500°C. Argon was the collision gas delivered from the gas bottle (Argon, 99.999%, AGA, Norway) with pressure in collision cell held at ~3.5 × 10–3 mbar. Table II presents MRM transitions and associated mass spectrometric parameters (cone voltage, collision energy and dwell time) for the compounds and the internal standards. System operation and data acquisition were controlled by the MassLynx V4.1 software (Waters Corp., Milford, USA). TargetLynx quantification program (Waters Corp., Milford, USA) was used to process all data. Method validation Procedures for method validation were performed as proposed by Peters et al. (17) and Rivier (18) including linearity, within- and between-assay precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), matrix effects (MEs), extraction recovery (RE), selectivity of the method and carry-over. Calibration curves and quantification Quantification of the samples was done by integrating the peak height of the specific MRM chromatogram relative to the integrated peak height of respective internal standard. Five-point calibration curves of second order (y = ax2 + bx + c), with a weighing factor of 1/x and the origin excluded, were used. Calibration curve fits and standard deviations were determined as a mean of the correlation coefficient (R2) of 10 between-assay calibrations and presented in Table I. Precision and accuracy QC samples at three different concentration levels were analyzed for each compound to evaluate between-day precisions (n = 10, expressed as RSD (%)), accuracies (expressed as biases (%)) and within-assay precisions (repeatability, n = 10, expressed as RSD %). The results are shown in Table I. LOD and LOQ LOD and LOQ (n = 10) were determined by the analysis of dilutions of the lowest calibrator in 10 independent assays. The acceptance criteria for LOD and LOQ were the followings: the signal-to-noise ratio should be ≥3 and ≥10 for both MRM transitions, respectively. For LOD: the ion ratios, calculated as a ratio between the integrated peak height of the quantifier and qualifier MRM transitions, should be ±20% from the mean ion ratio of calibrators and QC samples. For LOQ: the quantitative value should be within ±15% from the nominal value. Selectivity Selectivity of the method was investigated by analyzing stock solutions of the compounds important in forensic analysis that could be present in samples (opiates, amphetamines, benzodiazepines, z-hypnotics, antidepressants, antipsychotics, antiepileptic drugs and designer drugs). Interferences and signal overlap were evaluated for each compound. The results are presented in Supplementary Table 1. MEs, RE and carry-over MEs and RE were investigated for all compounds at three concentration levels by the approach proposed by Matuszewski et al. (19). Three sets of samples were analyzed. Set 1 was a neat calibrator, Set 2 a post-extraction spiked blood and Set 3 a pre-extraction spiked blood. MEs were defined as ME% = peak heights from the samples spiked after the sample preparation (Set 2) divided by the peak heights from the set of neat calibrators (Set 1). A value below 100 indicates ion suppression and a value above 100 indicates ion enhancement. A total of five different lots of blank blood, previously determined to be negative for cotinine and caffeine, were used. In addition, the MEs were evaluated in peripheral and central blood, pericardial fluid, vastus lateralis muscle, psoas muscle and vitreous humor (n = 6) at one concentration level (approved by the Regional committee for research Ethics in Norway, reference number 2012/2073). RE (RE%) was calculated as a ratio between the integrated peak heights from the samples spiked before the sample preparation (Set 3) and the integrated peak heights from the samples spiked after the sample preparation (Set 2). An additional calibrator with a concentration two times higher than the highest calibrator was prepared for the examination of possible carry-over. Figure 1. View largeDownload slide Ion chromatograms for the blank sample and the lowest calibrator. Figure 1. View largeDownload slide Ion chromatograms for the blank sample and the lowest calibrator. Results and Discussion Sample preparation A rapid sample preparation was accomplished with a simple protein precipitation of 100 μL of blood sample with 0.5 mL ice-cold acetonitrile/methanol mixture (85:15 v/v). The organic layer was evaporated to dryness and dissolved in the mobile phase. The recoveries were in the range 98–110% for cotinine, 101–104% for acetaminophen, 90–100% for ephedrine, 116–137% for caffeine, 103–108% for salicylic acid and 93–99% for dexchlorpheniramine (Table III). Table III. MEs, RE in blood and MEs in different media Compound  MEs in blood (n = 5)  Corrected MEs in different autopsy materialsa (n = 6)  QC sample concentration (mg/L)  ME (%)  RSD (%)  Corrected ME (%)  Extraction recovery (%)  QC sample conc. (mg/L)  1  2  3  4  5  6  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  Cotinine  0.00088  100  4.8  99  110  0.0035  96  7.4  98  3.9  93  4.7  119  17  119  23  98  0.2  0.0035  87  3.0  98  98  0.11  88  2.8  98  104  Acetaminophen  1.5  103  4.2  102  104  6.0  101  3.0  100  2.2  99  2.6  101  1.1  101  2.5  100  1.8  6.0  96  1.7  99  101  15  93  3.2  101  101  Ephedrine  0.061  99  7.7  107  90  0.24  89  8.1  86  7.3  88  4.1  89  4.7  90  5.5  94  4.4  0.24  86  7.2  98  95  0.61  92  7.8  102  100  Caffeine  0.39  141  17  138  118  1.6  110  7.4  108  5.9  107  7.7  106  5.2  110  8.3  117  10  1.6  95  5.7  107  116  3.9  96  9.2  108  137  Salicylic acid  1.4  131  9.0  109  103  5.5  127  1.7  125  4.4  125  2.5  126  2.1  131  2.4  126  2.6  5.5  109  2.4  96  108  14  94  1.7  100  105  Dexchlorpheniramine  0.020  81  3.8  101  99  0.078  101  2.1  99  5.1  96  4.4  91  7.0  93  12.1  103  2.4  0.078  75  3.0  103  94  0.20  84  3.2  111  93  Compound  MEs in blood (n = 5)  Corrected MEs in different autopsy materialsa (n = 6)  QC sample concentration (mg/L)  ME (%)  RSD (%)  Corrected ME (%)  Extraction recovery (%)  QC sample conc. (mg/L)  1  2  3  4  5  6  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  ME  RSD (%)  Cotinine  0.00088  100  4.8  99  110  0.0035  96  7.4  98  3.9  93  4.7  119  17  119  23  98  0.2  0.0035  87  3.0  98  98  0.11  88  2.8  98  104  Acetaminophen  1.5  103  4.2  102  104  6.0  101  3.0  100  2.2  99  2.6  101  1.1  101  2.5  100  1.8  6.0  96  1.7  99  101  15  93  3.2  101  101  Ephedrine  0.061  99  7.7  107  90  0.24  89  8.1  86  7.3  88  4.1  89  4.7  90  5.5  94  4.4  0.24  86  7.2  98  95  0.61  92  7.8  102  100  Caffeine  0.39  141  17  138  118  1.6  110  7.4  108  5.9  107  7.7  106  5.2  110  8.3  117  10  1.6  95  5.7  107  116  3.9  96  9.2  108  137  Salicylic acid  1.4  131  9.0  109  103  5.5  127  1.7  125  4.4  125  2.5  126  2.1  131  2.4  126  2.6  5.5  109  2.4  96  108  14  94  1.7  100  105  Dexchlorpheniramine  0.020  81  3.8  101  99  0.078  101  2.1  99  5.1  96  4.4  91  7.0  93  12.1  103  2.4  0.078  75  3.0  103  94  0.20  84  3.2  111  93  aDifferent media: 1 = peripheral blood, 2 = central blood, 3 = pericardial fluid, 4 = vastus lateralis muscle, 5 = psoas muscle and 6 = vitreous humor. Quantification and calibration curves A choice of preparing the blank samples, calibrators and the QC samples in mobile phase instead of in blood was made at the early stage of method development (Figure 1). Although the use of matrix-matched calibrators and samples is the ideal approach, the possibility to use water calibrators instead of whole blood calibrators has previously been demonstrated for several drugs of abuse (20). The use of dedicated isotope-labeled internal standards will, in addition, help to compensate for differences between the calibrators and samples. For salicylic acid and acetaminophen, proficiency testing has demonstrated the suitability of the method for analysis of whole blood. The fact that cotinine and specially caffeine were present in a large number of tested blank blood batches made this choice necessary to facilitate routine application of the method. The calibration curves (n = 10), created from five concentration levels, were found to be reproducible with RSDs ≤0.9% for all compounds in the given concentration ranges. Correlation coefficients were ≥0.995 for all the compounds. Calibration ranges, correlation coefficients R2 and RSDs of correlation coefficients are shown in Table I. Precision, accuracy, LOD and LOQ The within-assay precisions were between 1.1% and 15% and the between-assay precisions between 2.3% and 15%. The biases for the QC samples were within ±10% for all compounds, except for cotinine (14%) and dexchlorpheniramine (26%) at the lowest QC levels. The calculated precisions and biases together with LOD and LOQ values were found satisfactory (Table I). MEs, extraction recoveries and carry-over The residual matrix components still present after sample preparation have an influence on the MS ionization processes and may increase (ion enhancement) or decrease (ion suppression) the analytical signal (21). Five different blank blood batches and six different autopsy matrixes were used to determine the MEs. The results are presented in Table III. Deuterated internal standards were used for calculating the corrected MEs in blood. For all the compounds, the MEs were 100 ± 10% except caffeine (138%) and dexchlorpheniramine (111%). Slight ion suppression was observed for ephedrine in peripheral and central blood, pericardial fluid and vastus lateralis muscle from the autopsy cases. Cotinine showed ion enhancement in vastus lateralis and psoas muscle, and salicylic acid in all six autopsy matrixes. Extraction recoveries were found to be above 90% for all the compounds (Table III). Carry-over was investigated by analyzing a blank sample after a sample spiked to times higher than the highest calibrator. Carry-over was not observed for any of the compounds. Stability Stability experiments including freeze/thaw stability and long-term stability have not been performed. Several stability studies are available from the literature for cotinine (22–24), chlorpheniramine (dexchlorpheniramine is a dextrorotatory isomer of chlorpheniramine) (25), ephedrine (26–28), acetaminophen (29) and salicylic acid (30), indicating a good stability. Application of the method We have analyzed 119 samples from infants and children (≤4 years) during the period 2012–2016. Sixty-three cases (47%) were negative. Caffeine was detected solely in 15 (27%), cotinine in 32 (57%) and acetaminophen in 21 (38%) cases. Acetaminophen together with caffeine was found in two cases and together with dexchlorpheniramine in one case. Both acetaminophen and cotinine were present in two cases and caffeine and cotinine in five cases. Caffeine, cotinine and acetaminophen were proven in one case. Ephedrine and salicylic acid were not found in any of the samples. The data above are depersonalized data, so we do not know if there were other drugs present in these samples. Neither do we know the cause of deaths (i.e., SIDS, sudden unexpected death in childhood, accident). The samples received were varying: peripheral blood, central blood, thorax blood or fluid, blood from abdomen, blood from skull base, pericardial fluid, pleural effusion fluid and blood from hospital admission. Quantified concentrations are, therefore, not reported. Conclusions A fast, selective, and robust UHPLC–MS/MS method for the determination of acetaminophen, dexchlorpheniramine, caffeine, cotinine and salicylic acid in 100 μL of whole blood has been developed. The method was validated with the calibrator, control and blank samples prepared in the mobile phase instead of blood matrix because of caffeine and cotinine-free human blank blood is difficult to obtain. The deuterated internal standards coeluted with the compounds of interest, corrected MEs and compensated possible loses during the preparation of biological samples. 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Journal of Analytical ToxicologyOxford University Press

Published: Mar 1, 2018

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