Determination of lansoprazole in dog plasma using reversed phase - high performance liquid chromatography

Nội dung text: Determination of lansoprazole in dog plasma using reversed phase - high performance liquid chromatography

1. Journal of military pharmaco-medicine n o2-2018 DETERMINATION OF LANSOPRAZOLE IN DOG PLASMA USING REVERSED PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Luong Quang Anh*; Vu Ngoc Thang** Tran Nguyen Ha***; Nguyen Ngoc Chien*** SUMMARY Objectives: To determine lansoprazole (LPZ) in dog plasma. Methods: A single-step liquid- liquid extraction with tert-butyl methyl ether was used for LPZ extraction from plasma samples obtained. LPZ was determined by using reversed phase-high performance liquid chromatography (HPLC). Results: LPZ and internal standard were separated by using an isocratic mobile phase on reversed phase waters symmetry C18 column with the ultraviolet detection at 285 nm. Linearity range was 0.05 - 3.0 µg/mL (r > 0.999 of the regression line). The lower limitation of quantitation was 50 ng/mL. Mean percentage recoveries were from 97.67 to 101.02%. HPLC method was validated with intra-day and inter-day precision of 2.27 - 5.96% and 3.79 - 4.82%, respectively; accuracy was from 97.50 to 101.68%. LPZ was stable in plasma samples stored at four different degradation conditions. Conclusions: The method was successfully applied to determine LPZ in dog plasma samples. * Keywords: Lansoprazole; High performance liquid chromatography; Dog plasma; Validation. INTRODUCTION plasma. To estimate LPZ in plasma, some Lansoprazole, a proton pump inhibitor, studies revealed that HPLC method was reduces gastric acidity, an important factor necessary [1, 2, 3, 5]. However, there in treating acid-related disorders such as have been studies for determination of gastric ulcers, duodenal ulcers, and reflux LPZ in plasma in Vietnam up to now. oesophagitis. LPZ is used to treat gastro- Hence, we have studied to determine LPZ esophageal reflux, ulcers, acid-related in dog plasma corresponding with drug dyspepsia, etc [6]. LPZ is 97% bound to quality control requirements in fluids. This plasma proteins and shows its effectiveness research was also conducted to contribute correlating to drug concentration in object to further bioequivalence studies. * National Institute of Burns ** Military Institute of Pharmaceutical Analysis and Research *** Hanoi University of Pharmacy Corresponding author: Luong Quang Anh (luongquanganh@vmmu.edu.vn) Date received: 18/11/2017 Date accepted: 18/01/2018 89
2. Journal of military pharmaco-medicine n o2-2018 MATERIALS AND METHODS 5 µm) preceded by a steel guard column (RP18, 4 x 3 mm). The UV detector was 1. Materials and equipments. set at 285 nm. The flow-rate was 1 mL/min, Standard LPZ was provided from injection volume was 50 µL. The mobile National Institute of Drug Quality Control phase was a mixture of 65:35 (v/v) 0.01 M with its content of 99.5%. Pantoprazole potassium dihydrophosphate buffer solution (PPZ) as an internal standard was (pH was adjusted to 8.0 with triethylamine) provided from Institute of Drug Quality and ACN. Control in Hochiminh City with its content * Extraction of LPZ from plasma: of 93.87%. Methanol (MeOH), acetonitrile (ACN), triethylamine, tert-butyl methyl ether 50 µL internal standard solution of PPZ (TBME) were HPLC grade and purchased (40 µg/mL) and 2 mL tert butyl methyl from Merck (Germany) and all other ether were added to 500 µL of each ingredients used were of analytical grade. plasma sample. The solution was extracted Dog plasma was obtained from experimental by vortex-mixing for 3 minutes, followed healthy beagle dogs weighted from 10 to by centrifugation at 4,500 rpm/min for 0 12 kg following a standardized feeding 10 minutes at 20 C. A 1 mL aliquot of the process. supernatant obtained was transferred to a glass tube and evaporated until dry at 30 0C. Chromatographic measurements were The residue was dissolved in the solution made on the HPLC system (Shimadzu, containing 80 µL ACN and 120 µL 0.01 M Japan). The centrifuge machine (Eppendorf, potassium dihydro phosphate buffer Germany), vortex equipment (Heidolph, solution (Ph was adjusted to 8.0 with Germany) and vacuum dried centrifuge triethylamine) and mixed for 2 minutes. (miVAC, England) were used for LPZ and A 50 µL aliquot was subsequently injected PPZ extraction processes. All the equipments into the HPLC system. were validated yearly by applying the good laboratory practice criteria. * Validation method: The method was validated by application 2. Methods. the FDA guidelines [4] with respect to * Preparation of stock and standard specificity, linearity, accuracy and precision, solutions: extraction productivity, sample stability Stock solutions at concentration of and lower limit of quantitation (LLOQ). 1 mg/mL were prepared by dissolving a quantity of standard LPZ or internal RESULTS AND DISCUSSION standard in MeOH. Working standard 1. System suitability. solutions were diluted with MeOH when The HPLC system was equilibrated used for assay from prepared stock solutions. with the initial mobile phase composition, * Chromatographic conditions: followed by 6 injections of the same HPLC separation was carried out by standard solution including PPZ at 2 µg/mL using a RP18 steel column (150 x 4.6 mm; and LPZ at 1 µg/mL in mobile phase. 90
3. Journal of military pharmaco-medicine n o2-2018 Table 1: System suitability. PPZ LPZ Samples S t S t (min) F N R F N R (mAU.s) (min) (mAU.s) S ratios 1 4.123 215303 1.35 5123.69 8.193 131010 1.27 7894.50 0.6085 2 4.117 215303 1.36 5028.98 8.193 128443 1.19 7888.06 0.5966 3 4.117 215660 1.35 4949.28 8.193 131485 1.25 7847.23 0.6097 4 4.117 215208 1.33 5016.82 8.192 133088 1.27 7771.66 0.6184 5 4.120 215003 1.32 5053.88 8.193 131988 1.23 7845.69 0.6139 6 4.117 215773 1.33 4931.84 8.193 132293 1.21 7745.80 0.6131 Mean 4.12 8.19 0.6100 SD 0.0025 0.0004 0.01 %RSD 0.0609 0.0050 1.22 The system suitability parameters including peaks and theoretical plates met the chromatographic requirements. All parameters were satisfactory with good specificity for the stability assessment of PPZ and LPZ with good retention time (%RSD < 0.1) and S ratio (%RSD < 2). 2. LPZ extraction from plasma samples. Three methods for LPZ extraction from plasma samples were performed as follows: protein precipitation by MeOH (I); liquid-liquid extraction by a mixture of diethyl ether (DE) and dicloromethane (DCM) (70:30, v/v) (II); liquid-liquid extraction by TBME (III). Table 2: Productivities of different LPZ extraction methods from plasma samples. Concentration of 1.0 µg/mL Concentration of 2.0 µg/L Methods (n = 6) (n = 6) P (%) %RSD P (%) %RSD I 108.06 8.61 108.62 8.30 II 101.29 5.14 101.60 7.17 III 101.02 4.85 97.67 3.19 (Note: P = Mean of productivity). The results showed that all extraction methods from plasma samples had considerable productivity and stability. The liquid-liquid extraction by TBME showed more precision and reiteration than the two other methods. Besides, the representative chromatograms of LPZ and PPZ in extraction method using TBME was balanced and sharp without impurity peaks nearby the main peaks. The chromatograms also depicted straight background line and high LPZ peak. Thus, the liquid-liquid extraction by TBME has been chosen for LPZ isolation from obtained plasma samples. 91
4. Journal of military pharmaco-medicine n o2-2018 3. Validation method. - Specificity: Specificity is the ability to assess unequivocally the analyte in the presence of components that may be expected to be present in the sample matrix. For demonstrating the specificity of the method for drug assay in serum, the plasma samples were spiked (PPZ as an internal standard was added into blank plasma sample and LPZ plasma sample) and the representative chromatograms were shown on figure 1, figure 2 and figure 3. 0.04 0.03 mcAU 0.02 0.01 (Pantoprazol) (Lansoprazol) 0.00 0 1 2 3 4 5 6 7 8 9 10 Minutes Figure 1: Chromatogram of blank plasma sample. 0.04 0.03 0.02 mcAU 0.01 (Lansoprazol) 0.00 Pantoprazol 4.158 0 1 2 3 4 5 6 7 8 9 10 Minutes Figure 2: Chromatogram of blank plasma sample added by PPZ as an internal standard. 92
5. Journal of military pharmaco-medicine n o2-2018 0.04 0.03 0.02 mcAU 0.01 Lansoprazol 8.283 0.00 Pantoprazol 4.173 Pantoprazol 0 1 2 3 4 5 6 7 8 9 10 M inutes Figure 3: Chromatogram of LPZ plasma sample added by PPZ as an internal standard. The impurities did not interfere with the drug peak on figure 2 and figure 3, meanwhile, there were no impurity peaks from minutes 3 to minutes 10 on figure 1. The representative chromatograms on figure 2 and figure 3 showed that the PPZ and LPZ peaks were considerable, sharp and separately with its retention times of 4.1 and 8.2 minutes, respectively. Thus, the method is specific for PPZ and LPZ. * Linearity: The plasma samples were extracted to isolate LPZ and PPZ using the selected method. The residue was dissolved in mobile phase, then an amount of aliquot was subsequently injected into HPLC system. Peak area ratios between LPZ and PPZ were calculated. Calibration curves were performed on 6 different days and mean calibration curve was determined. Based on calibration curve, accuracy was calculated at each point of LPZ concentration. The results were shown in table 3. Table 3: Range and linearity. LPZ concentrations 0.05 0.1 0.2 0.5 1.0 1.5 2.0 3.0 (µg/mL) Peak area (PPZ) (mAU.s) 485904 482417 500250 488327 475907 477753 470826 450027 Peak area (LPZ) (mAU.s) 8601 15199 32382 77650 150307 217177 289451 415136 Peak area ratio (LPZ/PPZ) 0.0177 0.0315 0.0647 0.1590 0.3158 0.4546 0.6148 0.9225 Linear regression y = 0.3059x + 0.0033; R 2 = 0.9999 (y = ax + b) Calculated accuracy based 94.29 92.26 100.36 101.94 102.29 98.28 99.88 100.19 on calibration curve (%) %RSD of accuracy 17.14 14.66 9.38 4.56 2.78 3.06 3.18 1.53 (Notes : y = Peak area ratio; x = LPZ concentration (µg/mL) 93
6. Journal of military pharmaco-medicine n o2-2018 Linearity was established over the concentration range of 0.05 - 3.0 µg/mL for LPZ (0.05, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 3.0 µg/mL, respectively). Peak area ratios (y) of LPZ/PPZ were plotted versus LPZ respective concentrations (x) and linear regression analysis were performed on the resultant calibration curves. Coefficient correlation (R 2) was found to be more than 0.999 for all the analysis. Calculated accuracy was in range of 85 - 115% (from 92.26 to 102.29%). * Precision and repeatability: Precision and repetability assay was conducted on three samples as LQC, MQC and HQC containing LPZ following: 50 µL internal standard solution of PPZ was added to 500 µL of LPZ plasma samples at level of 0.2, 1.5, 2.5 µg/mL, then extracted by the chosen method. At each concentration, extraction of LPZ in plasma sample process was conducted in six replications on three consecutive days. LPZ concentration was determined by using calibration curve in the same analytical conditions. The data obtained from precise experiments were given in table 4 and table 5. Table 4: Intra-day precision and repeatability. LPZ found Precision Repeatability Samples (n = 6) LPZ added (µg/mL) (µg/mL) (%) (%RSD) LQC 0.2000 0.1950 97.50 5.96 MQC 1.5000 1.5228 101.52 3.03 HQC 2.4999 2.5421 101.68 2.27 Table 5: Inter-day precision and repeatability (3 days). LPZ found Precision Repeatability Samples (n = 18) LPZ added (µg/mL) (µg/mL) (%) (%RSD) LQC 0.2000 0.1968 98.40 4.82 MQC 1.5000 1.4931 99.54 3.79 HQC 2.4999 2.4995 99.98 3.87 In all three samples (LQC, MQC, HQC), intra-day and inter-day precisions were in range of 97.50 - 101.68%. The %RSD values of repeatability for the intra-day precision study and for the inter-day study were less than 10 confirming that the method was sufficiently precise for analytical requirements in fluids. * Lower limit of quantitation (LLOQ): LLOQ is the lowest amount analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. The LLOQ value was found to be 50 ng/mL for serum with the chromatogram depicted on figure 4 and figure 5. 94
7. Journal of military pharmaco-medicine n o2-2018 0.008 0.006 0.004 mcAU 0.002 Lansoprazol 8.237 0.000 Pantoprazol4.188 0 1 2 3 4 5 6 7 8 9 10 M inutes Figure 4: Chromatogram of LPZ at concentration 50 ng/mL. 0.008 0.006 0.004 mcAU 0.002 (Pantoprazol) (Lansoprazol) 0.000 -0.002 0 1 2 3 4 5 6 7 8 9 10 M inutes Figure 5: Chromatogram of blank serum . * LPZ stability studies: - Stability of LPZ in stock solution: Table 6: Stability of LPZ in stock solution. Stock LPZ concentration (mg/mL) Max difference solutions Day 0 Day 1 Day 3 Day 5 (%) N0 1 1.0250 1.0227 1.0236 1.0220 -0.29 N0 2 1.0400 1.0278 1.0265 1.0318 -1.30 N0 3 1.0150 1.0162 1.0142 1.0054 -0.95 Comparison of LPZ concentration in stock solution in MeOH after five days stored at 2 - 8 0C with LPZ concentration in immediate stock solution was used. At 1 st day, 3 rd day and 5 th day, LPZ in experimental stock solution was determined using the proposed method. The results showed that LPZ stock solutions were stable after storage at the experimental condition. 95
8. Journal of military pharmaco-medicine n o2-2018 - Stability of LPZ in serum: Table 7: Stability of LPZ in plasma samples. LPZ concentration in Actual LPZ degradation condition* Difference Stability studies concentration Mean ± SD Mean ± SD (%) (µg/mL, n = 6) (µg/mL, n = 6) Freeze-unfreeze process 0.1925 ± 0.0030 0.1918 ± 0.0016 -0.36 (3 periods) 2.0749 ± 0.0195 2.0151 ± 0.0376 -2.89 Stability after store at 0.1925 ± 0.0030 0.1898 ± 0.0050 -1.44 0 -45 C (30 days) 2.0749 ± 0.0195 2.0294 ± 0.0564 -2.20 Stability after store at room 0.1925 ± 0.0030 0.1922 ± 0.0056 -0.18 temperature (5 hours) 2.0749 ± 0.0195 2.0822 ± 0.0576 +0.35 Stability in autosampler 0.1925 ± 0.0030 0.1940 ± 0.0043 +0.75 0 (5 C, 8 hours) 2.0749 ± 0.0195 2.0845 ± 0.0168 +0.46 (*: LPZ concentration was calculated from calibration curve in the same analytical condition) Stability studies were established in plasma samples containing LPZ at concentration of 0.2 and 2.0 µg/mL. LPZ concentration of samples stored at different conditions was calculated and compared with that of initial correlative samples. The results showed that LPZ plasma samples were stable after storage at the experimental conditions. * Extraction productivity: - Extraction productivity of LPZ in serum: Table 8: Extraction productivity of LPZ in serum (n = 6). Concentration of 0.5 Concentration Concentration Parameters µg/mL of 1.0 µg/mL of 2.0 µg/mL Serum MP Serum MP Serum MP Mean of peak area (mAU.s) 42889 42817 136565 135183 265793 272128 % RSD 5.02 3.30 4.85 2.38 3.19 1.76 Productivity (%) 100.17 101.02 97.67 (Note: MP = Mobile phase) From the data in table 8, the selected extraction method performed high and stable productivity of LPZ in serum (from 97.67 to 101.02%, %RSD from 1.76 to 5.02). 96
9. Journal of military pharmaco-medicine n o2-2018 * Extraction productivity of PPZ as an internal standard: Assessment of PPZ extraction productivity at its concentration of 2 µg/mL was used. Table 9: Extraction productivity of PPZ in serum (n = 6). Peak area of PPZ in MP Peak area of PPZ in serum Productivity (%) Samples (mAU.s) (mAU.s) 1 470264 451093 95.92 2 478394 462837 96.74 3 473472 439982 92.92 4 475265 470018 98.89 5 479183 451004 94.11 6 479948 477257 99.43 Mean 476087.67 458698.50 96.35 %RSD 0.79 3.02 Mean extraction productivity of PPZ in serum was 96.35% and %RSD was 3.02. Hence, the selected extraction method was seen as an appropriate measure for determination of LPZ and PPZ internal standard from plasma samples. * Assay of commercial product: The validated method was applied to the determination of LPZ in plasma sample obtained from experimental healthy beagle dog used a commercial capsule containing 30 mg LPZ enteric coated pellets by oral route with single dose. The chromatogram of LPZ on figure 6 showed that the method was suitable for determination of LPZ in dog plasma. For this reason, it is possible to apply the method in bioequivalence studies of LPZ on experimental animal as dog. 0.02 0.00 Volts -0.02 Lansoprazol 67415 Pantoprazol424932 0 1 2 3 4 5 6 7 8 9 10 Minutes Figure 6: Chromatogram of LPZ and PPZ in plasma sample from beagle dog. 97
10. Journal of military pharmaco-medicine n o2-2018 CONCLUSION 4. FDA. Guidance for industry: Bioavailability The method for determination of LPZ and bioequivalence studies for orally in dog plasma has been established. The administered drug products - General advantages of the method were simple considerations. US Department of Health and mobile phase, suitable liquid-liquid Human Services. 2003, pp.1-23. extraction with high productivity. In 5. Karol M.D et al. Determination of LPZ addition, the method has good precision and five metabolites in plasma by HPLC. J and repeatability for application to Chromatogr B. 1995, 668, pp.182-186. determine LPZ in bioequivalence studies 6. Vietnam Ministry of Health. Vietnamese on experimental animals. national drug formulary. Medical edition. 2002. REFERENCES 7. Wu G.L et al. Determination of LPZ in human plasma by rapid resolution liquid 1. Aoki I et al. High-performance liquid chromatography-electrospray tandem mass chromatographic determination of LPZ and its spectrometry: Application to a bioequivalence metabolites in human serum and urine. J study on Chinese volunteers. J Pharm Biomed Chromatogr . 1991, 571, pp.283-290. Anal . 2008, 48, pp.1485-1489. 2. Borner K et al . Quantitative determination of LPZ in human serum by HPLC. 8. Zeinab A.E.S et al. Reversed-phase high Chromatographia . 1997, 45, pp.450-452. performance liquid chromatographic method 3. Dugger H.A et al. Bioequivalence for the determination of LPZ, omeprazole and evaluation of LPZ 30 mg capsules (Lanfast ® pantoprazole sodium sesquihydrate in presence and Lanzor ®) in healthy volunteers. Eur J of their acid-induced degradation products. Pharm Biopharm . 2001, 51 (2), pp.153-157. Chem Pharm Bull . 2006, 54 (6), pp.814-818. 98