TY - JOUR
AU - Andac, Sena, Caglar
AB - Abstract Dipeptidyl peptidase-4 inhibitors, so-called gliptins, constitute a fairly novel class of oral hypoglycemic agents. The development and validation of an automated online SPE-LC-UV method to determine intact sitagliptin, saxagliptin, vildagliptin and metformin simultaneously in human urine samples were performed. For the two-dimensional chromatographic separation, a Gemini C18 (250.0 × 4.6 mm i.d., 110 A0, 5.0 μ) analytical column and a gradient elution with 10.0 mM o-phosphoric acid and methanol and for the online SPE analysis of urine samples, a LiChrospher® ADS SPE-column (20.0 mm × 2.0 mm i.d., 25.0 μm) were used through the study. The fractionation, transfer, elution and separation of the spiked urine samples were achieved in just 9.57 min runtime with 12.0 mL of solvent consumption which was green and economical compared to other sample preparation methods. The calibration curves were determined to be linear in a wide range of 0.10–100.00 μg/mL with satisfactory regression coefficients. Method developed for two-dimensional determination of gliptins would be useful as a reference in therapeutic drug monitoring and screening for forensic medical cases which involve the abuse, unintentional or misuse of multiple gliptins in terms of its practical use, easy detection and reliable results. Introduction Type 2 diabetes mellitus (T2DM) represents a chronic metabolic disease resulting from functional insulin deficiency or insulin resistance, which may cause organ or function loss due to possible complications. Dipeptidyl peptidase-4 inhibitors (DPP-4), so-called gliptins, are fairly novel class of oral hypoglycemic agents and used for the treatment of T2DM (1). Sitagliptin (SITA), saxagliptin (SAX) and vildagliptin (VIL) are the most known members of this group of drugs (Figure 1). These medicines are available as stand-alone or as combined preparations with metformin (MET). DPP-4 is an extracellular (expressed on cell-surface or released into blood stream) proteolytic enzyme that cleaves proteins from the N-terminus of dipeptide sequences X-ala and X-pro. It is indeed one of the volatile exopeptidases that can hydrolyze hard-to-cleave X-pro sequences due to shielding effect (2). The main physiological role of DPP-4 is to regulate blood glucose level by cleaving the active form of glucagon-like peptide (GLP-1) into an inactive form, which then turns-off insulin secretion to prevent excessive downregulation of blood sugar level (2). DPP-4 inhibitors function to increase intact GLP-1 levels to continue insulin secretion in a high blood-glucose-level-dependent manner in patients with T2DM (2–4), lowering the risks of hypoglycemia and weight gain (5). DPP-4 inhibitors on market, such as SITA, SAX and VIL are applied in patients with T2DM either as a monotherapy along with diet and exercise or as add-on combination therapy along with MET, sulfonylureas, thiazolidinediones and sodium–glucose cotransporter-2 inhibitors (6). Figure 1 Open in new tabDownload slide Chemical structures of SITA, SAX, VIL and MET. Figure 1 Open in new tabDownload slide Chemical structures of SITA, SAX, VIL and MET. Table I The time program for the left and right pumps, where A is methanol, B is aqueous solution of 10.0 mM o-phosphoric acid and C is water . STEP . Time (min) . Valve position . Pump 1 . Pump 2 . % B . % C . Flow (mL/min) . % B . % C . Flow (mL/min) . Fractionation 1 0.00 0 100 0 - 0 95 1.0 2 1.55 0 100 0 - 0 95 1.0 Transfer/Elution 3 1.56 1 100 0 1.0 0 0 - 4 4.50 1 10 0 1.0 0 0 - 5 6.50 1 35 0 1.0 0 0 - 6 7.50 1 45 0 1.0 0 0 - 7 8.55 1 45 0 1.0 0 95 1.0 Wash/Conditioning 8 8.56 0 100 0 1.0 0 10 1.0 9 9.56 0 100 0 1.0 0 10 1.0 10 9.57 0 100 0 1.0 0 95 1.0 . STEP . Time (min) . Valve position . Pump 1 . Pump 2 . % B . % C . Flow (mL/min) . % B . % C . Flow (mL/min) . Fractionation 1 0.00 0 100 0 - 0 95 1.0 2 1.55 0 100 0 - 0 95 1.0 Transfer/Elution 3 1.56 1 100 0 1.0 0 0 - 4 4.50 1 10 0 1.0 0 0 - 5 6.50 1 35 0 1.0 0 0 - 6 7.50 1 45 0 1.0 0 0 - 7 8.55 1 45 0 1.0 0 95 1.0 Wash/Conditioning 8 8.56 0 100 0 1.0 0 10 1.0 9 9.56 0 100 0 1.0 0 10 1.0 10 9.57 0 100 0 1.0 0 95 1.0 Open in new tab Table I The time program for the left and right pumps, where A is methanol, B is aqueous solution of 10.0 mM o-phosphoric acid and C is water . STEP . Time (min) . Valve position . Pump 1 . Pump 2 . % B . % C . Flow (mL/min) . % B . % C . Flow (mL/min) . Fractionation 1 0.00 0 100 0 - 0 95 1.0 2 1.55 0 100 0 - 0 95 1.0 Transfer/Elution 3 1.56 1 100 0 1.0 0 0 - 4 4.50 1 10 0 1.0 0 0 - 5 6.50 1 35 0 1.0 0 0 - 6 7.50 1 45 0 1.0 0 0 - 7 8.55 1 45 0 1.0 0 95 1.0 Wash/Conditioning 8 8.56 0 100 0 1.0 0 10 1.0 9 9.56 0 100 0 1.0 0 10 1.0 10 9.57 0 100 0 1.0 0 95 1.0 . STEP . Time (min) . Valve position . Pump 1 . Pump 2 . % B . % C . Flow (mL/min) . % B . % C . Flow (mL/min) . Fractionation 1 0.00 0 100 0 - 0 95 1.0 2 1.55 0 100 0 - 0 95 1.0 Transfer/Elution 3 1.56 1 100 0 1.0 0 0 - 4 4.50 1 10 0 1.0 0 0 - 5 6.50 1 35 0 1.0 0 0 - 6 7.50 1 45 0 1.0 0 0 - 7 8.55 1 45 0 1.0 0 95 1.0 Wash/Conditioning 8 8.56 0 100 0 1.0 0 10 1.0 9 9.56 0 100 0 1.0 0 10 1.0 10 9.57 0 100 0 1.0 0 95 1.0 Open in new tab Sample preparation methods, i.e. liquid–liquid extraction (LLE) or solid-phase extraction (SPE), are typically conducted off-line, performed either semiautomatically or manually. These techniques utilize large volumes of solvents and consumables, increasing toxicity risk, cost and labor. Despite the fact that the amount of solvent volume has been severely reduced by micro-extraction processes, such as solid-phase micro-extraction (SPME) (7) or liquid/liquid micro-extraction (8), the automation of such methods to liquid-chromatographic systems may not be as managable and affordable. Methods of direct online injection reduce the steps regarding preparation, enabling the efficient clean-up and preconcentration of biological fluids, thereby minimizing the possible usage of toxic chemicals, cost, labor and time (9). Restricted-access materials (RAMs) are biocompatible materials used when preparing samples, allowing the direct injection of biological fluids. A special class of materials is represented by these sorbents and can separate the matrix of protein and the analyte fraction in a biological specimen relying on a molecular-weight cutoff. In two-dimensional liquid chromatography (2D-LC), the RAM column acts as a pre-column for the analytical column. The first dimension was achieved by RAM as size-exclusion by the selective removal of the high molecular weight matrix components of urine samples, while the second dimension was achieved by the selective extraction of low molecular weight target analytes with compounds that are retained by partition in a selective manner, ion exchange, and/or adsorption, via a chemical or physical diffusion barrier. Alkyl-diol silica (ADS) material represents the most commonly used RAM, having a structure as externally glyceryl-propyl or diol groups and internally butyryl (C4), capryloyl (C8) or stearoyl (C18) groups, allowing various applications in the pretreatment of samples (10, 11). Table II System suitability data . Retention time (min) . Resolution . Plates . Tailing factor . S/N ratio . SITA 6.95 5.44 179496 1.10 113.80 SAX 6.77 3.01 213539 1.08 63.50 VIL 6.36 6.39 132369 1.33 19.00 MET 4.06 31.17 42363 1.66 98.60 . Retention time (min) . Resolution . Plates . Tailing factor . S/N ratio . SITA 6.95 5.44 179496 1.10 113.80 SAX 6.77 3.01 213539 1.08 63.50 VIL 6.36 6.39 132369 1.33 19.00 MET 4.06 31.17 42363 1.66 98.60 (n = 3). Open in new tab Table II System suitability data . Retention time (min) . Resolution . Plates . Tailing factor . S/N ratio . SITA 6.95 5.44 179496 1.10 113.80 SAX 6.77 3.01 213539 1.08 63.50 VIL 6.36 6.39 132369 1.33 19.00 MET 4.06 31.17 42363 1.66 98.60 . Retention time (min) . Resolution . Plates . Tailing factor . S/N ratio . SITA 6.95 5.44 179496 1.10 113.80 SAX 6.77 3.01 213539 1.08 63.50 VIL 6.36 6.39 132369 1.33 19.00 MET 4.06 31.17 42363 1.66 98.60 (n = 3). Open in new tab Table III The range, calibration curve equation, regression coefficient, LOD, LOQ and recovery values for SITA, SAX, VIL and MET . Concentration range (μg/mL) . Calibration curve (y = ax + b) ± SD . Regression coefficient (R2) ± SD . Recovery (%) ± SD . LOD (μg/mL) . LOQ (μg/mL) . SITA 0.10–100.00 y = (0.4952 ± 0.01) x-(0.4149 ± 0.02) 0.9989 ± 0.01 96.7 ± 0.02 0.017 0.058 SAX 0.10–100.00 y = (0.2388 ± 0.01)x + (0.1008 ± 0.01) 0.9993 ± 0.01 99.8 ± 0.05 0.025 0.083 VIL 0.10–100.00 y = (0.1043 ± 0.02)x + (0.0855 ± 0.01) 0.9988 ± 0.02 98.8 ± 0.04 0.035 0.078 MET 0.10–100.00 y = (0.5578 ± 0.01)x-(0.9749 ± 0.01) 0.9946 ± 0.01 96.3 ± 0.02 0.025 0.083 . Concentration range (μg/mL) . Calibration curve (y = ax + b) ± SD . Regression coefficient (R2) ± SD . Recovery (%) ± SD . LOD (μg/mL) . LOQ (μg/mL) . SITA 0.10–100.00 y = (0.4952 ± 0.01) x-(0.4149 ± 0.02) 0.9989 ± 0.01 96.7 ± 0.02 0.017 0.058 SAX 0.10–100.00 y = (0.2388 ± 0.01)x + (0.1008 ± 0.01) 0.9993 ± 0.01 99.8 ± 0.05 0.025 0.083 VIL 0.10–100.00 y = (0.1043 ± 0.02)x + (0.0855 ± 0.01) 0.9988 ± 0.02 98.8 ± 0.04 0.035 0.078 MET 0.10–100.00 y = (0.5578 ± 0.01)x-(0.9749 ± 0.01) 0.9946 ± 0.01 96.3 ± 0.02 0.025 0.083 (n = 3). Open in new tab Table III The range, calibration curve equation, regression coefficient, LOD, LOQ and recovery values for SITA, SAX, VIL and MET . Concentration range (μg/mL) . Calibration curve (y = ax + b) ± SD . Regression coefficient (R2) ± SD . Recovery (%) ± SD . LOD (μg/mL) . LOQ (μg/mL) . SITA 0.10–100.00 y = (0.4952 ± 0.01) x-(0.4149 ± 0.02) 0.9989 ± 0.01 96.7 ± 0.02 0.017 0.058 SAX 0.10–100.00 y = (0.2388 ± 0.01)x + (0.1008 ± 0.01) 0.9993 ± 0.01 99.8 ± 0.05 0.025 0.083 VIL 0.10–100.00 y = (0.1043 ± 0.02)x + (0.0855 ± 0.01) 0.9988 ± 0.02 98.8 ± 0.04 0.035 0.078 MET 0.10–100.00 y = (0.5578 ± 0.01)x-(0.9749 ± 0.01) 0.9946 ± 0.01 96.3 ± 0.02 0.025 0.083 . Concentration range (μg/mL) . Calibration curve (y = ax + b) ± SD . Regression coefficient (R2) ± SD . Recovery (%) ± SD . LOD (μg/mL) . LOQ (μg/mL) . SITA 0.10–100.00 y = (0.4952 ± 0.01) x-(0.4149 ± 0.02) 0.9989 ± 0.01 96.7 ± 0.02 0.017 0.058 SAX 0.10–100.00 y = (0.2388 ± 0.01)x + (0.1008 ± 0.01) 0.9993 ± 0.01 99.8 ± 0.05 0.025 0.083 VIL 0.10–100.00 y = (0.1043 ± 0.02)x + (0.0855 ± 0.01) 0.9988 ± 0.02 98.8 ± 0.04 0.035 0.078 MET 0.10–100.00 y = (0.5578 ± 0.01)x-(0.9749 ± 0.01) 0.9946 ± 0.01 96.3 ± 0.02 0.025 0.083 (n = 3). Open in new tab Table IV Intra-day and inter-day precision findings at three various concentrations for SITA, SAX, VIL and MET in the spiked urine sample . Spiked concentration (μg/mL) . Calculated concentration (μg/mL) ± SD . Repeatability (for inter-day) RSD % . Intermediate precision (for intra-day) RSD % . F-values from ANOVA analysis (FEXP < FCRITICAL, %95) . SITA 0.25 0.26 ± 0.02 7.93 8.42 1.12 < 5.14 2.50 2.49 ± 0.03 1.13 1.74 1.38 < 5.14 50.00 49.98 ± 2.92 5.98 5.99 0.01 < 5.14 SAX 0.25 0.25 ± 0.01 3.46 7.09 3.20 < 5.14 2.50 2.45 ± 0.15 0.27 0.40 1.12 < 5.14 50.00 50.01 ± 0.17 3.37 6.16 2.33 < 5.14 VIL 0.25 0.24 ± 0.01 3.55 4.54 0.63 < 5.14 2.50 2.49 ± 0.01 7.29 10.26 0.98 < 5.14 50.00 50.72 ± 1.05 0.38 0.38 0.01 < 5.14 MET 0.25 0.25 ± 0.02 7.37 7.41 0.01 < 5.14 2.50 2.51 ± 0.25 9.61 9.67 0.01 < 5.14 50.00 49.97 ± 0.35 0.67 0.73 0.17 < 5.14 . Spiked concentration (μg/mL) . Calculated concentration (μg/mL) ± SD . Repeatability (for inter-day) RSD % . Intermediate precision (for intra-day) RSD % . F-values from ANOVA analysis (FEXP < FCRITICAL, %95) . SITA 0.25 0.26 ± 0.02 7.93 8.42 1.12 < 5.14 2.50 2.49 ± 0.03 1.13 1.74 1.38 < 5.14 50.00 49.98 ± 2.92 5.98 5.99 0.01 < 5.14 SAX 0.25 0.25 ± 0.01 3.46 7.09 3.20 < 5.14 2.50 2.45 ± 0.15 0.27 0.40 1.12 < 5.14 50.00 50.01 ± 0.17 3.37 6.16 2.33 < 5.14 VIL 0.25 0.24 ± 0.01 3.55 4.54 0.63 < 5.14 2.50 2.49 ± 0.01 7.29 10.26 0.98 < 5.14 50.00 50.72 ± 1.05 0.38 0.38 0.01 < 5.14 MET 0.25 0.25 ± 0.02 7.37 7.41 0.01 < 5.14 2.50 2.51 ± 0.25 9.61 9.67 0.01 < 5.14 50.00 49.97 ± 0.35 0.67 0.73 0.17 < 5.14 (n = 3). Open in new tab Table IV Intra-day and inter-day precision findings at three various concentrations for SITA, SAX, VIL and MET in the spiked urine sample . Spiked concentration (μg/mL) . Calculated concentration (μg/mL) ± SD . Repeatability (for inter-day) RSD % . Intermediate precision (for intra-day) RSD % . F-values from ANOVA analysis (FEXP < FCRITICAL, %95) . SITA 0.25 0.26 ± 0.02 7.93 8.42 1.12 < 5.14 2.50 2.49 ± 0.03 1.13 1.74 1.38 < 5.14 50.00 49.98 ± 2.92 5.98 5.99 0.01 < 5.14 SAX 0.25 0.25 ± 0.01 3.46 7.09 3.20 < 5.14 2.50 2.45 ± 0.15 0.27 0.40 1.12 < 5.14 50.00 50.01 ± 0.17 3.37 6.16 2.33 < 5.14 VIL 0.25 0.24 ± 0.01 3.55 4.54 0.63 < 5.14 2.50 2.49 ± 0.01 7.29 10.26 0.98 < 5.14 50.00 50.72 ± 1.05 0.38 0.38 0.01 < 5.14 MET 0.25 0.25 ± 0.02 7.37 7.41 0.01 < 5.14 2.50 2.51 ± 0.25 9.61 9.67 0.01 < 5.14 50.00 49.97 ± 0.35 0.67 0.73 0.17 < 5.14 . Spiked concentration (μg/mL) . Calculated concentration (μg/mL) ± SD . Repeatability (for inter-day) RSD % . Intermediate precision (for intra-day) RSD % . F-values from ANOVA analysis (FEXP < FCRITICAL, %95) . SITA 0.25 0.26 ± 0.02 7.93 8.42 1.12 < 5.14 2.50 2.49 ± 0.03 1.13 1.74 1.38 < 5.14 50.00 49.98 ± 2.92 5.98 5.99 0.01 < 5.14 SAX 0.25 0.25 ± 0.01 3.46 7.09 3.20 < 5.14 2.50 2.45 ± 0.15 0.27 0.40 1.12 < 5.14 50.00 50.01 ± 0.17 3.37 6.16 2.33 < 5.14 VIL 0.25 0.24 ± 0.01 3.55 4.54 0.63 < 5.14 2.50 2.49 ± 0.01 7.29 10.26 0.98 < 5.14 50.00 50.72 ± 1.05 0.38 0.38 0.01 < 5.14 MET 0.25 0.25 ± 0.02 7.37 7.41 0.01 < 5.14 2.50 2.51 ± 0.25 9.61 9.67 0.01 < 5.14 50.00 49.97 ± 0.35 0.67 0.73 0.17 < 5.14 (n = 3). Open in new tab Despite the fact that there is a number of analytical methods to determine gliptins in biological fluids in the literature as a single drug or when combined with MET (6, 12–17), there is no simultaneous determination method available for SITA, SAX, VIL and MET in urine with LC-UV. There are two chromatographic methods for the simultaneous determination of SITA, SAX, VIL and MET, which have been developed with MS/MS detection only in plasma samples. In one of them, saxagliptin was not included (17), and in the other one, sample preparation was carried out by LLE, and SITA, SAX and VIL were not quantified, they were only identified with this method (18). The developed and validated method in this study may be the first fully automated online SPE-LC-UV method to determine SITA, SAX, VIL and MET simultaneously in human urine samples, ensuring that spiked human urine samples are repetitively injected in a direct way. It can be applied in clinical laboratories in terms of its practical use, easily found detection and reliable results requiring no sample preparation. Experimental Reagents and solutions Sitagliptin phosphate monohydrate (SITA) was kindly supplied by Merck Sharp and Dohme Pharmaceuticals (NJ, USA). Metformin (MET) and vildagliptin (VIL) were kindly provided by Abdi Ibrahim Ilac (Istanbul, Turkey), whereas saxagliptin (SAX) was obtained from Richem International Co., Ltd. (Shanghai, China), internal standard (Doxazosin; IS) was purchased from Sigma-Aldrich (Steinheim, Germany). Ortho-phosphoric acid (o-PA) (≥98%) and methanol (HPLC grade) were purchased from Merck KGaA (Darmstadt, Germany). An Elga water purification system (London, UK) was utilized for the purification of deionized water, reaching a resistivity of 18.2 MΩ. The preparation of stock solutions that contained SITA, SAX, VIL, MET and IS was carried out in water, and the samples were further diluted with water. All the solutions were stored at a temperature of +4°C. Figure 2 Open in new tabDownload slide Schematic configuration of the fully automated online SPE-LC-PDA system, where A is the initial valve position for fractionation, B is the switched valve position for transfer, P1 and P2 are the pumps, AS is the autosampler, V is the valve, AC is the analytical column, D is the detector and W is the waste. Figure 2 Open in new tabDownload slide Schematic configuration of the fully automated online SPE-LC-PDA system, where A is the initial valve position for fractionation, B is the switched valve position for transfer, P1 and P2 are the pumps, AS is the autosampler, V is the valve, AC is the analytical column, D is the detector and W is the waste. Figure 3 Open in new tabDownload slide A chromatogram of a spiked urine sample with 50.0 μg/mL concentration of SITA, SAX, VIL and MET and 25.0 μg/mL of IS compared to a blank urine chromatogram at 212 nm. Figure 3 Open in new tabDownload slide A chromatogram of a spiked urine sample with 50.0 μg/mL concentration of SITA, SAX, VIL and MET and 25.0 μg/mL of IS compared to a blank urine chromatogram at 212 nm. Figure 4 Open in new tabDownload slide A chromatogram of a spiked urine sample with 100.0 μg/mL concentration of SITA (4), SAX (3), VIL (2) and MET (1) and 25.0 μg/mL of IS (5) with 250, 237, 225 and 212 nm wavelengths. Figure 4 Open in new tabDownload slide A chromatogram of a spiked urine sample with 100.0 μg/mL concentration of SITA (4), SAX (3), VIL (2) and MET (1) and 25.0 μg/mL of IS (5) with 250, 237, 225 and 212 nm wavelengths. Preparation of standard solutions SITA, SAX, VIL and MET were prepared in 1.0 mg/mL concentration as a main stock mixture. The preparation of working solutions was carried out as a result of the dilution of the main stock solution with water to a concentration of 100.0, 10.0, and 1.0 μg/mL, respectively. The preparation of the IS stock solution was prepared in 1.00 mg/mL concentration, further diluted to 100.00 μg/mL with water, subsequently 25.00 μL of this solution was spiked to all of the sample solutions. Sample preparation After collecting the raw urine sample from a healthy volunteer, samples were diluted 10-fold; dilution of samples was performed using water. During the analyses, 20.0 μL of the urine sample was introduced to the online SPE-LC-PDA system. Drug-free urine samples were prepared as nine calibration standards for SITA, SAX, VIL and MET to yield concentrations between 0.10 and 100.00 μg/mL. Three levels of urine quality controls (0.25, 2.50 and 50.00 μg/mL) were studied to assess inter-day and intra-day precision and accuracy. To prepare solutions with 100.00, 50.00, 10.00 and 5.00 μg/mL concentrations, 100 μL of the urine test sample was first spiked with 100.00, 50.00, 10.00 and 5.00 μg/mL of 1.0 mg/mL main stock solution, respectively, and 25.00 μg/mL of the IS stock solution, vortexed, and completed to 1.00 mL with water. The same procedure was used to prepare samples with concentrations of 2.50, 1.00 and 0.50 μg/mL by spiking 25.00, 10.00 and 5.00 μL of 100.00 μg/mL stock solution, respectively, and 25.00 μg/mL of the IS stock solution to 100.00 μL of the urine sample, following which they were vortexed and completed to 1.00 mL with water. To prepare solutions with 0.25 and 0.10 μg/mL concentrations, 100.00 μL of the urine test was first spiked with 25.00 and 10.00 μL of 10.00 μg/mL main stock solution, respectively, and 25.00 μg/mL of the IS stock solution, vortexed, and completed to 1.00 mL with water. Each sample was prepared in three replicates and injected three times directly to the online SPE-LC-PDA system with a volume of 20.00 μL. Spiked urine sample was compared with blank urine sample. Chromatographic conditions and online SPE-LC setup LC-grade methanol and aqueous solution of 10.00 mM o-PA solution were used as mobile phases A and B, respectively, and 10.00 mM o-PA solution was prepared by solving 70.00 μL of the o-PA solution in 100.00 mL of ultrapure water. Both mobile phases were degassed in an ultrasonic bath prior to use. Analyses were examined by utilizing a completely automated online SPE-LC-PDA system, the scheme of which was presented in Figure 2. A Thermo Scientific Dionex Ultimate 3000 Rapid Separation LC framework with Chromeleon 6.8 software was utilized. The device comprises a dual gradient pump, an autosampler, a PDA Detector, a 10-Port switching valve and a column oven. The PDA was adjusted to 212, 225, 237 and 250 nm, and 1 mL/min of the flow rate was used for all the fractionation, conditioning, separation, and elution steps. A Gemini C18, (250.0 × 4.6 mm i.d., 110 A0, 5.0 μ) analytical column was used for separation. The gradient elution program (Table I) was operated for a period of 9.57 min. The online SPE was executed on a self-packed LiChrospher® ADS RP 4 RAM SPE-column (20.0 mm × 2.0 mm ID, 25.0 μm; Merck KGaA, Darmstadt, Germany) to fractionate urine samples. Pump 1 was used for transfer and elution while pump 2 was used for sample fractionation, SPE wash and conditioning. The time program for the pumps is shown in Table I, where A is methanol, B is aqueous solution of 10.0 mM o-PA and C is water. The determination and calculation of the valve switching parameters tM (the time to deplete the sample matrix completely), tA (the breakthrough time of the analyte) and tT (the desorption/transfer time of the analyte from an SPE-column to an analytical column) were performed, as described by Majors et al. (11). Method validation In order to validate the method, the linearity and range, precision, accuracy, selectivity, the limit of detection (LOD) and the limit of quantification (LOQ) parameters were studied based on the ICH Guidelines (19). Selectivity was proved through performing separations with satisfactory resolutions between the peaks. Specifity was assessed by injecting blank urine samples subsequent to repetitive injection of spiked urine samples at the end of each sequence thus any possible carry-over issue was evaluated as well. To verify that the system would perform in accordance with the criteria set forth in the procedure, retention time, resolution, tailing factor, signal-to-noise ratio and theoretical plate number values of the analytes were examined as a part of system suitability data. Linearity and range were studied by constructing the calibration curves with nine plots in the spiked urine samples between the concentrations of 0.10 and 100.00 μg/mL for SITA, SAX, VIL and MET. The peak area ratios of the analyte to the IS were plotted versus the analyte concentration. To assess the accuracy and inter-day and intra-day precision, the concentrations of 0.25, 2.50 and 50.00 μg/mL of SITA, SAX, VIL and MET in urine were analyzed. The inter-day precision was studied in three different days in the same pattern. The one-way analysis of variance (ANOVA) analysis was conducted to measure the inter-day and intra-day precision (repeatability) at three various levels (n = 3). Excel software was used to perform the ANOVA test. At a 95% confidence level, experimental F and critical F were compared. Recovery was studied as a result of the comparison of the online SPE-LC analysis of a spiked urine sample with the calibration standard prepared in water. Furthermore, the off-line recovery of the drugs was obtained by injecting the samples directly into the analytical column. The formula of x × s/slope of the calibration curve was used to find LOD and LOQ, x = 10 for LOD and x = 3 for LOQ, where s value is the SD of the regression lines. The carry-over of the online sample preparation system was assessed as a result of the injection of the blank urine sample following a high-concentration spiked urine sample. Results Method validation Some of the system suitability data of the developed method are given in Table II. The functionality of the chromatographic system was verified with satisfactory system suitability data having resolution > 2, tailing factor < 2, theoretical plate number > 2000 and signal-to-noise ratio > 10. The calibration curves were determined to be linear in a wide range between 0.10 and 100.0 μg/mL with satisfactory regression coefficients. Maximum 15% RSD values were used for calculations through the validation study. It was determined that the LOD and LOQ values were satisfactory as the urine excretion was taken into account which was discussed in detail in application to human urine samples. The inter-day and intra-day precision were studied on low, medium and high concentrations by using Excel software to perform the ANOVA test. The recovery of the online SPE-LC-PDA method was compared with the result of the same level standard solution injection to the analytical column and was found to be between 99.3 and 90.0%. Specifity and selectivity The fully automated online SPE-LC-PDA system ensures that biological samples are repetitively injected into the chromatographic system in a direct way without a need for further sample pretreatment. For this purpose, a RAM SPE-column packed with LiChrospher® ADS RP4 material was utilized to fractionate urine samples in a size-selective manner throughout the study. As could be seen in Figure 3, it was ensured that the urine sample was repetitively injected into the system with no carry-over issue, which was proved by injecting a blank urine sample at the end of each sequence. The method was proved to be specific regarding the absence of any interaction on the Rt of the analyte peaks. Well-resolved analyte peaks were eluted between 4.00 and 7.35 min, where the retention times were 4.06, 6.36, 6.77, 6.95 and 7.32 min for MET, VIL, SAX, SIT and IS, respectively. The chromatograms were obtained setting the PDA detector to several wavelengths as 212, 225, 237 and 250 nm, and 212 nm was chosen due to its better sensitivity (Figure 4). Selectivity was proved with the well-resoluted peaks for each of the analyte and the absence of any interaction in the rt of each analyte arising from an other analyte when the analytes are given alone in the injections. Linearity and calibration The calibration curves were found to be linear between 0.10 and 100.00 μg/mL for SITA SAX, VIL and MET. The concentration range, calibration curve equation, regression coefficient, LOD, LOQ and recovery values for SITA, SAX, VIL and MET are shown in Table III. The introduced method was revealed to be linear in a wide range of concentrations with satisfactory regression coefficients for the mentioned drugs. Precision, accuracy, LOD and LOQ The LOD and LOQ values were determined to be 0.02–0.08 μg/mL for SAX, 0.04–0.12 μg/mL for VIL, 0.02–0.06 μg/mL for SITA and 0.02–0.08 μg/mL for MET, respectively. The inter-day and intra-day precision were studied on low, medium and high concentrations, which were 0.25, 2.50 and 50.00 μg/mL for SITA, SAX, VIL and MET. The inter-day precision was studied in three different days. The one-way ANOVA analysis was conducted to measure the inter-day and intra-day precision (repeatability) at three various levels (n = 3). Excel software was used to perform the ANOVA test. At a 95% confidence level, experimental F and critical F were assessed comparing the spiked solutions in Table IV. The recovery of the online SPE-LC-PDA method was compared with the result of the same level standard solution injection into the analytical column and found to be 96.7, 99.8, 98.8 and 96.3% for SITA, SAX, VIL and MET, respectively, as shown in Table III. Discussion Optimization of chromatographic conditions Separation on the LC column was investigated first by configuring the system as a direct LC. The standard solutions were first directly injected into the analytical column. Since the drugs of interest have basic pKa values and polar character owing to the addition of amino and cyano groups, the reversed-phase chromatographic separation was tried with acidic solutions, ACN and MeOH mobile phase combinations and the best resolution was obtained with a MeOH/10.0 mM o-PA mobile phase system with gradient elution on a Gemini C18 LC column. This mobile phase combination was also good enough to elute our polar analytes from RP4 size exclusion column resulting high yield of recovery over 90%. Optimization of the online SPE-LC method The online SPE technique ensuring the direct injection of biological specimens into chromatography eliminates sample pretreatment, decreases the analysis time and meets the automation and high-throughput analysis needs. Online SPE-LC is a fully automated sample preparation method, in which sample preparation and separation are combined. After achieving the separation on the LC column with a direct injection, the system was converted into an online SPE-LC configuration. Afterward, the size exclusion was achieved with a self-packed LiChrospher® ADS RP4 RAM SPE-column (20.0 mm × 2.0 mm i.d., 25.0 μm), on which the drug substances were retained, but high molecular weight matrix compounds, such as proteins, nucleic acids, etc. were excluded. Urine samples were fractionated with a mobile phase mixture of H2O/MeOH: 95/5, v/v for 1.5 min by coupling the SPE column directly to the system, as illustrated in Figure 2 as the initial valve position. After the depletion of all the high molecular weight matrix compounds successfully, the SPE column was coupled to the LC column as an online SPE-LC configuration. In the subsequent introduction of spiked urine samples to the online system, the runtime of analysis was 8.5 min, where the first 1.5 min were for matrix depletion, the next 7.0 min were for transfer and separation and 1.0 min was for wash and reconditioning. Preparation and separation of urine samples was achieved with 12.0 mL of solvent consumption on a repetitively usable SPE column. In one run, 5.9 mL of methanol (49.4%), 4.3 mL of 10.0 mM o-PA (35.8%), 1.8 mL of water (14.8%) and, in the whole validation study, 1080.0 mL of the solvent was consumed. Application to human urine samples Daily urine excretion of a healthy person was reported on average to be 1971.1 ± 690.7 mL/day (20). The renal clearance (CLrenal) of VLG was published to be 13 ± 2.3 L/h (22), which was about 32% of its total clearance (CLtotal = 40.6 ± 8.97 L/h) (23). 23% of orally administered 50 mg VLG was eliminated in intact form by the kidneys (24) and elimination half time (t½ (h)) was reported as 2.13 ± 0.722. 50-mg of orally administered VLG gave rise to 5.75 mg of intact VLG in urine. An average volume of urine sample corresponding to the elimination half time of VLG was equaled to 174.94 mL, yielding a VLG urine concentration of 32.87 μg/mL. Similarly, 80% of intact STG administered orally (100 mg) was excreted by renal elimination (25, 26). An average fraction recovered in urine in 24 h for STG is given as 0.53 (27) which corresponds 21.15 mg of STG yielding 10.75 μg/mL urine concentration. In the absorption, distribution, metabolism and excretion (ADME) study of SXG, ~75% of the total radioactive dose of SXG was recovered in the urine (comprising 24% SAX) and t½ (h) was reported as 3.09 ± 0.65 (28, 29). 5-mg of orally administered SAX gave rise to 0.6 mg of intact SAX in urine. An average volume of urine sample corresponding to the elimination half time of SAX was equaled to 253.78 mL, yielding a SAX urine concentration of 2.36 μg/mL. It was determined that the LOD and LOQ values were satisfactory as the urine concentrations during excretion was taken into account. Conclusion Method developed in this study was directly applied to human urine samples to which the gliptins were spiked at varying concentrations, which involves noninvasive sampling as compared to invasive blood sampling. Preparation and separation of spiked urine samples was achieved on a repetitively usable SPE column with a green and affordable consumption of the solvents. Sample preparation was achieved automatically by online SPE coupled with LC-UV to determine SITA, SAX and VIL simultaneously when MET was present in spiked human urine samples for the first time, and the validation of the developed method was carried out based on the ICH guidelines. The 2D-LC method developed here to determine multiple gliptins in urine samples could be well utilized in the determination of an unknown sample of urine obtained from a patient by overdose or misuse of an unknown gliptin type antidiabetic drug. In that respect, the facile HPLC method for the determination of gliptins in urine samples linearly covered a wide range of concentrations which allowed cross-referencing the in- and out-of-range laboratory results. Acknowledgement This research was approved ethically by Local Ethics Committee of Istanbul University, Institute of Cardiology, with an approval number of B.08.06. YOK. 2. I.U. E. 50. 0. 05. 00/02, on 5 March 2014. A part of this study was presented as an oral presentation at the Euroanalysis XX Conference held on 1–5 September 2019, in Istanbul, Turkey. Funding This work was funded by Scientific Research Projects Coordination Unit of Istanbul University with Project number: BEK-2017-26400. References 1. Bohannon , N. ; Overview of the gliptin class (dipeptidyl peptidase-4 inhibitors) in clinical practice ; Postgraduate Medicine , ( 2009 ); 121 : 40 – 45 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Drucker , D.J. , Nauck , M.A.; The incretin system: Glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes ; Lancet , ( 2006 ); 368 : 1696 – 1705 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Deacon , C.F. , Mannucci , E., Ahren , B.; Glycaemic efficacy of glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors as add-on therapy to metformin in subjects with type 2 diabetes-a review and meta analysis ; Diabetes Obesity Metabolism , ( 2012 ); 14 : 762 – 767 . Google Scholar Crossref Search ADS WorldCat 4. Henry , R.R. , Smith , S.R., Schwartz , S.L.; Effects of saxagliptin on β-cell stimulation and insulin secretion in patients with type 2 diabetes ; Diabetes Obesity Metabolism , ( 2011 ); 13 : 850 – 858 . Google Scholar Crossref Search ADS WorldCat 5. Liu , S.C. , Tu , Y.K., Chien , M.N.; Effect of antidiabetic agents added to metformin on glycaemic control, hypoglycaemia and weight change in patients with type 2 diabetes: A network meta-analysis ; Diabetes Obesity Metabolism , ( 2012 ); 14 : 810 – 820 . Google Scholar Crossref Search ADS WorldCat 6. Frederich , R. , McNeill , R., Berglind , N.; The efficacy and safety of the dipeptidyl peptidase-4 inhibitor saxagliptin in treatment-naïve patients with type 2 diabetes mellitus: A randomized controlled trial ; Diabetes Obesity Metabolism , ( 2012 ); 24 : 36 – 39 . Google Scholar OpenURL Placeholder Text WorldCat 7. Ma , M. , Cantwell , F.F.; Solvent microextraction with simultaneous back-extraction for sample cleanup and preconcentration: Quantitative extraction ; Analytical Chemistry , ( 1998 ); 70 : 3912 – 3919 . Google Scholar Crossref Search ADS WorldCat 8. Boos , K.-S. , Rudolph , A., Vielhauer , S., Walfort , A., Lubda , D., Eisenbeiss , F.; Alkyl-diol silica (ADS): Restricted-access precolumn packings for direct injection and coupled-column chromatography of biofluids ; Fresenius' Journal of Analytical Chemistry , ( 1995 ); 352 : 684 – 690 . Google Scholar Crossref Search ADS WorldCat 9. Hermansson , J. , Grahn , A.; Determination of drugs by direct injection of plasma into a biocompatible extraction column based on a proteinentrapped hydrophobic phase ; Journal of Chromatography A , ( 1994 ); 660 : 119 – 129 . Google Scholar Crossref Search ADS PubMed WorldCat 10. Gonçalves , H.D. , Abrão , L.C.C., Gonçalves , M., Adriano , S., Barbosa , F., Figueiredo , E.C.; New advances in restricted access materials for sample preparation: A review ; Analytica Chimica Acta , ( 2017 ); 959 : 43 – 65 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Majors , R.E. , Boos , K.S., Grimm , C.H., Lubda , D., Wieland , G.; Practical guidelines for HPLC-integrated sample preparation using column switching ; LC-GC Separation Science , ( 1996 ); 14 : 554 – 560 . Google Scholar OpenURL Placeholder Text WorldCat 12. Fura , A. , Khanna , A., Vyas , V.; Pharmacokinetics of the dipeptidyl peptidase 4 inhibitor saxagliptin in rats, dogs, and monkeys and clinical projections ; Drug Metabolism and Disposition , ( 2009 ); 37 : 1164 – 1171 . Google Scholar Crossref Search ADS PubMed WorldCat 13. El-Zaher , A.A. , Hashem , H.A., Elkady , E.F.A.; A validated LC-MS/MS bioanalytical method for the simultaneous determination of dapagliflozin or saxagliptin with metformin in human plasma ; Microchemical Journal , ( 2019 ); 149 : 113 – 118 . Google Scholar Crossref Search ADS WorldCat 14. Zeng , W. , Musson , D.G., Fisher , A.L., Alison , L.; Determination of sitagliptin in human urine and hemodialysate using turbulent flow online extraction and tandem mass spectrometry ; Journal of Pharmaceutical and Biomedical Analysis , ( 2008 ); 46 : 534 – 542 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Barseem , A. , Hytham , A., El-Shabrawy , Y.; The use of SDS micelles as additive to increase fluorescence analysis of sitagliptin and saxagliptin derivatives in their tablets and human plasma ; Microchemical Journal , ( 2019 ); 146 : 20 – 24 . Google Scholar Crossref Search ADS WorldCat 16. Deshpande , P.B. , Butle , S.R.; Determination of dipeptidyl peptidase-4 inhibitors by spectrophotometric and chromatographic methods ; Journal of Analytical Chemistry , ( 2018 ); 73 : 303 – 316 . Google Scholar Crossref Search ADS WorldCat 17. Al Bratty , M. , Alhazmi , H.A., Javed , S.A.; Development and validation of LC-MS/MS method for simultaneous determination of metformin and four gliptins in human plasma ; Chromatographia , ( 2017 ); 80 : 891 – 899 . Google Scholar Crossref Search ADS WorldCat 18. Hess , C. , Musshoff , F., Madea , B.; Simultaneous identification and validated quantification of 11 oral hypoglycaemic drugs in plasma by electrospray ionisation liquid chromatography-mass spectrometry ; Analytical and Bioanalytical Chemistry , ( 2011 ); 400 : 33 – 41 . Google Scholar Crossref Search ADS PubMed WorldCat 19. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use ICH Harmonised Tripartite Guideline Validation of Analytical Procedures ; Text And Methodology Q2(R1) . ( 2005 ), https://database.ich.org/sites/default/files/Q2_R1__Guideline.pdf. (accessed March 23, 2020) . 20. Perinpam , M. , Ware , E.B., Smith , J.A., Turner , S.T., Kardia , S.L.R., Perinpam , J.C.L.; Key influence of sex on urine volume and osmolality ; Biology of Sex Differences , ( 2016 ); 7 : 12 – 18 . Google Scholar Crossref Search ADS PubMed WorldCat 21. Center for Drug Evaluation and Research, U.S. Food and Drug Administration ; Reviewer Guidance, Validation of Chromatographic Methods . FDA , Rockville, MD , ( 1994 ). Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 22. Deacon , C.F. , Nauck , M.A., Toft-Nielsen , M.; Both subcutaneously and intravenously administered glucagon-like peptide I are rapidly degraded from the NH2-terminus in type II diabetic patients and in healthy subjects ; Diabetes , ( 1995 ); 44 : 1126 – 1131 . Google Scholar Crossref Search ADS PubMed WorldCat 23. Baetta , R. , Corsini , A.; Pharmacology of dipeptidyl peptidase-4 inhibitors: Similarities and differences ; Drugs , ( 2011 ); 71 : 1441 – 1467 . Google Scholar Crossref Search ADS PubMed WorldCat 24. He , Y.L. ; Clinical pharmacokinetics and pharmacodynamics of vildagliptin ; Clinical Pharmacokinetics , ( 2012 ); 51 : 147 – 162 . Google Scholar Crossref Search ADS PubMed WorldCat 25. Migoya , E.M. , Stevens , C.H., Bergman , A.J., Luo , W., Lasseter , K.C., Dilzer , S.C. et al. ; Effect of moderate hepatic insufficiency on the pharmacokinetics of sitagliptin ; Canadian Journal of Clinical Pharmcology , ( 2009 ); 16 : 165 – 170 . Google Scholar OpenURL Placeholder Text WorldCat 26. Sekar , R. , Singh , K., Arokiaraj , A.W.R., Chow , B.K.C.; Pharmacological actions of glucagon-like peptide-1, gastric inhibitory polypeptide, and glucagon ; International Review Cell Molecular Biology , ( 2016 ); 326 : 279 – 341 . Google Scholar Crossref Search ADS WorldCat 27. Januvia (sitagliptin) Procedure No. EMEA/H/C/000722/P46 030 CHMP assessment report for paediatric use studies submitted according to Article 46 of the Regulation (EC) No 1901/2006 . https://www.ema.europa.eu/en/documents/variation-report/januvia-h-c-722-p46-0030-epar-assessment-report_en.pdf ( accessed March 23, 2020 ). 28. Boulton , D. , Frevert , L.L.E.U., Tang , A., Castaneda , L., Vachharajani , N.N., Kornhauser , D.M. et al. ; Influence of renal or hepatic impairment on the pharmacokinetics of saxagliptin ; Clinical Pharmacokinetics , ( 2011 ); 50 : 253 – 265 . Google Scholar Crossref Search ADS PubMed WorldCat 29. Onglyza™ (saxagliptin) : US Prescribing Information . https://www.accessdata.fda.gov/drugsatfda_docs/label/2015/022350s016lbl.pdf ( accessed March 23, 2020 ) © The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Cross Referencing 2D-LC Determination of Intact Gliptins in Urine
JO - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmaa059
DA - 2020-10-26
UR - https://www.deepdyve.com/lp/oxford-university-press/cross-referencing-2d-lc-determination-of-intact-gliptins-in-urine-0x9mk91Ay3
SP - 907
EP - 914
VL - 58
IS - 10
DP - DeepDyve
ER -