To validate the determination method of nifedipine (NIF) in dog plasma by ultra performance liquid chromatography with tandem mass spectrometric detection (UPLC-MS/MS). The evaluation of the specificity, selectivity, lower limit of quantitation, accuracy, precision, calibration curve and the linear range, recovery, carry-over, matrix effect, stability of NIF in plasma following the guidance of US-FDA.
Journal of military pharmaco-medicine no5-2020 STUDY ON DETERMINATION OF NIFEDIPINE IN DOG PLASMA BY ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY WITH TANDEM MASS SPECTROMETRIC DETECTION Tran Quang Trung1, Nguyen Thanh Hai2 Trinh Van Lau3, Nguyen Huu Duy1, Nguyen Thi Dao4 SUMMARY Objectives: To validate the determination method of nifedipine (NIF) in dog plasma by ultra performance liquid chromatography with tandem mass spectrometric detection (UPLC-MS/MS) Methods: The evaluation of the specificity, selectivity, lower limit of quantitation, accuracy, precision, calibration curve and the linear range, recovery, carry-over, matrix effect, stability of NIF in plasma following the guidance of US-FDA Results: The UPLC-MS/MS method has been evaluated and achieved the US-FDA’s regulations Conclusion: The UPLC-MS/MS method can be used to determine the NIF in dog plasma, which will be used in bioavailability studies of the NIF dosage forms * Keywords: Nifedipine; UPLC-MS/MS; Dog plasma INTRODUCTION Nifedipine (NIF), a calcium channel blocker, is the first dihydropyridine drug to be clinically used to treat coronary venous insufficiency in 1969 [2] It is widely used in the treatment of angina, hypertension and other vascular disorders [1, 3, 4] According to the Biopharmaceutics Classification System (BCS), NIF is a typical Class II compound with high permeability, lipid solubility, which is rapidly and completely absorbed from the gastrointestinal tract after oral administration It has a very low bioavailability of 45 - 75%, mainly due to pre-systemic metabolism, which may result in very low plasma concentrations [1] Several methods for determination of NIF in plasma samples by gas chromatography, voltammetric method or HPLC with UV detection and electrochemical detection have been reported [5, 6, 7, 8, 9] Training Institute of Pharmacy, Vietnam Military Medical University School of Medicine and Pharmacy, Vietnam National University, Hanoi National Institute of Drug Quality Control Thanh Phat Pharmacy Joint Stock Company Corresponding author: Tran Quang Trung (tqt201316@gmail.com) Date received: 29/6/2020 Date accepted: 06/7/2020 97 Journal of military pharmaco-medicine no5-2020 However, these methods are not sensitive enough to determine NIF in low plasma concentrations In order to conduct the bioavailability studies of the NIF dosage forms by rapid, highly sensitive and accurate method, based on existing equipments, especially with very low plasma NIF concentration, we conducted this study aiming: To determine NIF in dog plasma by UPLC-MS/MS MATERIALS AND METHODS Materials and equipments * Materials, solvents and chemicals: - Standards: Reference standard of NIF (potency 99.82%) was provided by National Institute of Drug Quality Control Control Number: WS 0216200.02 Internal standard of Glibenclamid (potency 100.01%) was provided by National Institute of Drug Quality Control Control Number: 0103129, moisture content: 0.09% - Solvents and chemicals: * Equipments: All analytical equipments meet ISO/IEC 17025 - 2005 and GLP standard, including: Quantum Ultra Triple Quadrupole Mass Spectrometer system Alliance Waters, Detector Xevo TQD (USA), electrospray ionization (ESI) source; Hypersil Gold column (C18, 1.8 µm, 50 x 2.1 mm); Analytical balance Mettler Toledo (precision 0.01 mg); Elmasonic S100H ultrasonic shaker (Germany); HS 260 horizontal shaker (IKA, Germany); Genious vortex shaker (IKA, Germany); deep fridge (Panasonic, Japan) and other common instruments of appropriate accuracy in laboratory Methods * Chromatographic conditions: - Analytical device: UPLC-MS/MS system (Water) - Chromatographic column: C18; 50 x 2.1 mm; 1.9 µm Column temperature: 400C - Mobile phase: MeCN: formic acid 0.1% (90 : 10, v/v) HPLC grade acetonitrile, methanol, acid formic were procured from Merck Ltd - Flow rate: 0.2 mL/min Analytical grade chloroform was procured from Merck Ltd - Injection size: µL - Blank plasma: Dog plasma without analytical substance were supplied by Animal Board, Vietnam Military Medical University Lactose monohydrate, sodium chloride, magnesium stearate, PVP K30, red iron oxide were sourced from China and all meet USP 38 standards 98 - Detector: Xevo TQD - Autosampler temperature: 200C * Mass conditions: + Mass spectrometry type: MS/MS, electrospray ionization source ESI (+) + Parameters of mass spectrometry device for NIF and glibenclamid detection are presented in the table Journal of military pharmaco-medicine no5-2020 Table 1: Parameters of mass spectrometry detector for qualitative and quantitative NIF and internal standard glibenclamid Analytical substance Nifedipin IS (Glibenclamid) ESI (+) ESI (+) Capillary voltage (kV) 4 Cone voltage (V) 24 34 Desolvation temperature ( C) 350 350 Desolvation gas flow (L/H) 850 850 Cone gas flow (L/H) 20 20 Collision energy (V) 14 Parent ion (m/z) 347.07 494.20 Product ion (m/z) 315.02 369.12 Parameters Ionization mode * Sample preparation: nitrogen stream Dissolve the residue in Allow the plasma sample to thaw at 0.5 mL of mobile phase and inject into the room temperature Take sufficiently 0.5 UPLC-MS/MS system mL of the plasma sample, transfer it into a * Method validation: 15 mL glass vial and add exactly 50 µL of internal standard solution (glibenclamide solution of 0.4 µg/mL) It was vortexed for seconds and extracted with mL of chloroform for minutes in a horizontal shaker at 300 times/minute Samples were centrifuged at 4,000 rpm for minutes Take exactly mL of the organic phase into a 15 mL glass vial and evaporated without heat to dry under The proposed UPLC-MS/MS method of NIF analysis was validated to assess fundamental parameters such as: Specificity, selectivity, lower limit of quantitation, accuracy, precision, calibration curve and the linear range, recovery, carry-over, matrix effect, stability of NIF in plasma, according to the recommendations of the current United State Food and Drug Administration (U.S FDA) [10] 99 Journal of military pharmaco-medicine no5-2020 RESULTS AND DISSCUSION Specificity and selectivity DHCL-BLANK4 Smooth(Mn,1x2) 100 % 0.01 0.18 0.44 MRM of channels,ES+ 347.07 > 315.02 1.469e+003 nifedipin 0.74 104 nifedipin 0.74 104 1.15 1.27 DHCL-BLANK4 Smooth(Mn,1x2) 100 MRM of channels,ES+ 494.20 > 369.12 3.312e+002 Glibenclamid 0.74 39 Glibenclamid;0.74;39 % 0.01 0.18 1.40 1.03 0.20 0.40 0.60 0.80 1.00 1.15 1.20 1.40 Figure 1: Chromatogram of blank plasma sample DHCL-LLOQ4 Smooth(Mn,1x2) MRM of channels,ES+ 347.07 > 315.02 2.672e+004 nifedipin 0.74 1717 100 % DHCL-LLOQ4 Smooth(Mn,1x2) MRM of channels,ES+ 494.20 > 369.12 2.230e+005 Glibenclamid 0.75 13639 100 % 0.20 0.40 0.60 0.80 1.00 1.20 1.40 Figure 2: Chromatogram of spiked sample containing reference standard of NIF and internal standard of glibenclamide at LLOQ concentration of 0.5 ng/mL and concentration of 40 ng/mL, respectively Analyze the blank plasma and spiked samples containing reference standard of NIF at a concentration of about 0.5 ng/mL and internal standard of glibenclamide following the developed method The results showed that peak area response of blank plasma sample at the retention time corresponding to that of NIF (0.74 minutes) was 7% smaller than peak area response of NIF in concentration of 0.5 ng/mL, respectively and peak area response of blank plasma sample at the retention time corresponding to that of GLI (0.75 minutes) did not exceed 1% of peak area response of GLI Hence, the procedure is specific and selective to NIF and GLI in accordance with the regulations of bioanalytical method 100 Journal of military pharmaco-medicine no5-2020 Calibration curve and the linear range Table 2: The linearity of the method obtained after regression analysis CC1 CC CC2 Ratio CC3 Ratio CC4 Ratio CC5 Conc (ng/mL) NIF/GLI Conc (ng/mL) NIF/GLI Conc (ng/mL) NIF/GLI Conc (ng/mL) NIF/GLI Ratio Conc (ng/mL) NIF/GLI Ratio S1 0.5 0.116 0.5 0.131 0.5 0.141 0.5 0.132 0.5 0.135 S2 1.0 0.227 1.0 0.234 1.0 0.267 1.0 0.25 1.0 0.26 S3 2.0 0.426 2.0 0.435 2.0 0.507 2.0 0.501 2.0 0.487 S4 5.0 1.020 5.0 1.056 5.0 1.225 5.0 1.161 5.0 1.178 S5 10.0 1.981 10.0 2.117 10.0 2.357 10.0 2.285 10.0 2.385 S6 20.1 4.122 20.1 4.214 20.1 4.674 20.0 4.751 20.1 4.678 S7 40.1 7.681 40.1 7.984 40.1 8.930 40.1 8.803 40.2 8.983 S8 80.2 15.343 80.2 16.259 80.2 18.005 80.2 17.786 80.3 18.144 S9 100.3 19.857 100.3 20.676 100.3 22.186 100.2 23.299 100.4 23.044 a 0.1980 0.2047 0.2298 0.2291 0.2303 b 0.0198 0.0287 0.0300 0.0193 0.0223 r 0.9994 0.9998 0.9993 0.9994 0.9997 (Conc.: Concentration) Table 3: Accuracy of standard samples Precision compared to the theoretical concentration (%) CC CC1 CC2 CC3 CC4 CC5 S1 97.1 99.8 97.0 98.4 98.1 S2 104.5 100.5 103.3 100.8 103.1 S3 102.5 99.2 103.7 105.2 100.9 S4 101.0 100.4 104.0 99.7 100.4 S5 99.0 102.1 101.3 98.9 102.6 S6 103.1 101.7 100.5 103.3 100.6 S7 96.5 96.9 96.6 95.6 96.8 S8 96.5 98.9 97.5 96.7 98.0 S9 99.9 100.6 96.1 101.4 99.6 101 Journal of military pharmaco-medicine no5-2020 Plasma samples containing reference standard of NIF were prepared at concentrations from 0.5 - 100 ng/mL Analyze the samples following the developed method The correlation between the concentration of NIF (x) and the peak area ratio of NIF and GLI (y) has been studied by linear regression method, used weighting coefficient (1/concentration2) The results indicated that a linear correlation between the concentration of NIF and the peak area ratio of NIF and GLI was obtained in the range of concentrations from 0.5 - 100 ng/mL with the correlation coefficient r > 0.99 The concentrations of NIF determined from the calibration curve compared with the theoretical concentrations were within the accepted limits in accordance with the regulations of bioanalytical method (80 - 120% for the lowest concentration, 85 - 115% for the remaining concentrations) Lower limit of quantitation Table 4: Validation results of lower quantitative limits of the method Concentrations of NIF determined from the calibration curve (ng/mL) Compared to the actual concentration (%) Signal to noise ratio (S/N) 0.6 113.5 > 10 0.5 100.4 > 10 0.6 112.2 > 10 0.6 119.5 > 10 0.6 123.8 > 10 Sample Theoretical concentrations (ng/mL) 0.5 Mean (%) 113.9 CV (%) 7.8 Analyze the blank plasma and spiked samples containing reference standard of NIF at an exact concentration of approximately 0.5 ng/mL and internal standard of glibenclamide (LLOQ sample) The concentration of NIF in LLOQ samples was determined from the calibration curve, which was carried out in parallel under the same conditions The results showed that S/N value of NIF peak in LLOQ samples were greater than 10 Mean ratios of NIF concentration in the samples which were determined from the calibration curve compared with the theoretical concentrations were within the range of 80 - 120%, which indicates that the results met the requirements of the lower quantitative limit of bioanalytical method following the guidance of US-FDA 102 Journal of military pharmaco-medicine no5-2020 Accuracy and precision Table 5: Validation results of intra-day precision and accuracy Sample LLOQ LQC HQC HQC (0.5 ng/mL) (1.5 ng/mL) ( 50.2 ng/mL) (75.3 ng/mL) Conc (ng/mL) (a) Accuracy (b) (%) 0.6 Conc (ng/mL) (a) Accuracy (b) (%) 113.5 1.6 0.5 100.4 0.6 Conc (ng/mL) (a) Accuracy (b) (%) 105.6 43.1 1.6 109.4 112.2 1.7 0.6 119.5 0.6 Mean CV (%) Conc (ng/mL) (a) Accuracy (b) (%) 85.8 71.9 95.5 49.6 98.9 70.9 94.2 116.2 51.1 101.8 67.7 89.9 1.6 108.5 50.0 99.5 70.9 94.2 123.8 1.6 106.6 48.4 96.5 71.2 94.6 0.6 113.9 1.6 109.3 48.4 96.5 70.5 93.7 7.7 7.8 2.8 3.8 6.5 6.5 2.3 2.3 (Conc.: Concentration) Table 6: Validation results of inter-day precision and accuracy Sample LLOQ LQC HQC HQC (0.5 ng/mL) (1.5 ng/mL) ( 50.2 ng/mL) (75.3 ng/mL) Conc (ng/mL) Day Day (a) Accuracy (b) (%) 0.6 Conc (ng/mL) (a) Accuracy (b) (%) 113.5 1.6 0.5 100.4 0.6 Conc (ng/mL) (a) Accuracy (b) (%) 105.6 43.1 1.6 109.4 112.2 1.7 0.6 119.5 0.6 Conc (ng/mL) (a) Accuracy (b) (%) 85.8 71.9 95.5 49.6 98.9 70.9 94.2 116.2 51.1 101.8 67.7 89.9 1.6 108.5 50.0 99.5 70.9 94.2 123.8 1.6 106.6 48.4 96.5 71.2 94.6 0.5 97.7 1.6 106.6 46.9 93.4 74.8 99.3 0.5 100.2 1.5 100.2 48.1 95.8 70.7 93.9 0.5 107.7 1.6 107.8 49.7 98.9 68.2 90.6 0.5 107.7 1.7 110.1 49.5 98.5 70.5 93.6 0.6 119.1 1.5 102.4 48.0 95.6 70.3 93.4 103 Journal of military pharmaco-medicine no5-2020 0.5 106.5 1.5 103.0 42.4 84.5 67.3 89.4 0.6 110.4 1.5 98.2 44.7 89.1 65.2 86.6 0.5 108.9 1.4 96.4 43.1 85.8 62.4 82.8 0.4 83.4 1.4 91.5 44.2 88.1 63.7 84.7 0.5 101.8 1.4 94.0 39.9 79.4 65.2 86.6 Mean 0.5 107.5 1.5 103.8 46.6 92.8 68.7 91.3 CV (%) 11.6 9.4 6.4 6.5 7.3 7,3 5,0 5.0 Day (Conc.: Concentration) Intra-day or inter-day precision and accuracy were validated by five replicate analysis of LLOQ, LQC, MQC and HQC samples containing NIF at concentrations of 0.5; 1.5; 50; 75 ng/mL, respectively The content of NIF in the samples was determined from the calibration curve and the percentage ratio between the concentration determined from the calibration curve and the theoretical concentration The results indicated that mean accuracy for intra-day and inter-day batch ranged from 93.7 - 113.9% for NIF and 91.3 - 107.5% for NIF, respectively Similarly, intra-day and inter-day precisions ranged from 2.3 - 7.8% and 5.0 - 11.6% for NIF, which indicates that the results met the requirements of the intra-day or inter-day precision and accuracy of bioanalytical method following the guidance of US-FDA (≤ 20% at LLOQ and ≤ 15% for others) Recovery Table 7: Results of recovery percentage of NIF and GLI Recovery percentage (%) NIF LQC 104.7 MQC 99.0 HQC 106.6 GLI 104.3 Recovery percentage of NIF and GLI were determined by comparing the mean peak area of NIF and GLI obtained by injecting five extracted samples of LQC, MQC and HQC with the mean peak area obtained by injection of respective aqueous standard solutions in blank matrix The results showed that sample preparation method with high and stable extraction efficiency (the difference between concentrations is not more than 10%) 104 Journal of military pharmaco-medicine no5-2020 Matrix effect Table 8: Results of matrix effect MFNIF Sample MFGLI MFNIF/MFGLI LQC HQC LQC HQC LQC HQC 0.513 0.536 0.659 0.594 0.779 0.903 0.564 0.395 0.658 0.463 0.858 0.853 0.543 0.490 0.610 0.576 0.891 0.851 0.497 0.440 0.554 0.530 0.897 0.831 0.454 0.507 0.577 0.499 0.787 1.015 0.805 0.555 0.726 0.605 1.109 0.917 Mean 0.887 0.895 CV (%) 13.5 7.5 Collect blank plasma samples of various origins Extract the blank plasma samples according to the developed procedure for obtaining the respective sample matrix solutions Prepare the standards at concentrations of LQC and HQC in sample matrix solutions in parallel with the standards at these in mobile phase Matrix effect was evaluated through MFNIF/MFIS ratio which was determined by comparing NIF area response and GLI area response of LQC and HQC in sample matrix solutions with NIF area response and GLI area response of these in mobile phase The results showed that there was no difference between the different sample matrix with CV < 15% Carry-over Table 9: Results of carry-over Blank plasma sample LLOQ sample Sample Blank plasma sample/ Mean LLOQ Conclusion NIF GLI NIF GLI NIF GLI 110 13 1374 10699 0,080 0,0012 Qualified 165 42 1374 10536 0,119 0,0040 Qualified 133 40 1377 10610 0,096 0,0038 Qualified 153 35 1319 10526 0,111 0,0033 Qualified 76 1461 10366 0,055 0,0005 Qualified 108 0,078 0,0000 Qualified Mean 1381 10547 105 Journal of military pharmaco-medicine no5-2020 Prepare blank plasma samples, LLOQ samples and ULOQ samples containing reference standard of NIF at a concentration of about 0.5 ng/mL, 100 ng/mL following the developed method Inject the blank plasma samples after each ULOQ sample in UPLC-MS/MS system The results indicated that peak area response of blank plasma sample at the retention time corresponding to that of NIF was 20% smaller than peak area response of NIF in LLOQ concentration of 0.5 ng/mL and peak area response of blank plasma sample at the retention time corresponding to that of IS was 5% smaller than peak area response of IS in LLOQ concentration of 0.5 ng/mL, which reveals that the results met the requirements of carry-over of bioanalytical method in accordance with the guidance of US-FDA Stability of NIF in plasma Table 10: Results of stability of NIF in plasma Sample Theoretical concentration (ng/mL) Concentration after storage (ng/mL; n = 3) Difference LQC 1.5 1.66 10.7 HQC 75.3 64.0 -15.0 Short-term stability LQC 1.5 1.54 2.6 (5 hour; room temparature) HQC 75.3 64.0 -15.0 Autosampler stability LQC 1.5 1.63 8.7 (24 hour/4 C) HQC 75.3 68.98 -8.4 Long - term stability LQC 1.5 1.57 4.8 HQC 75.3 68.37 -9.2 Stability Three freeze-thaw cycles stability 0 (%) (-60 C ± -5 C, 45 days) Stability of NIF in plasma was investigated using spiked samples at two different concentration levels (LQC, HQC) prepared in triplicate Stability of NIF in plasma was evaluated by preparing the concentration of NIF in storage samples with theoretical concetration The results showed that concentrations of NIF in the LQC and HQC samples that were stored for three freeze-thaw cycles, five hours in room temparature, 24 hours in autosampler and 45 days in temperature of -60 ± 50C differs from the theoretical concentration by not more than 15% Therefore, NIF was stable in plasma under the above storage conditions 106 Journal of military pharmaco-medicine no5-2020 CONCLUSION A rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry method was developed and validated to determine NIF in dog plasma with a lower limit of quantification of 0.5 ng/mL The method has a wide linear range, high accuracy, precision with CV < 15%, quick and simple sample preparation method It can be applied to determine NIF in bioavalbility studies of the NIF dosage forms REFERENCES Bộ Y tế Dược thư Quốc gia Việt Nam Nhà xuất Y học Hà Nội 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