Quantitative determination of acyclovir in dogs’ plasma by high performance liquid chromatography spectrometry

Journal of military pharmaco-medicine n o 1-2020 121 QUANTITATIVE DETERMINATION OF ACYCLOVIR IN DOGS’ PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY SPECTROMETRY Le Van Thanh1; Vu Thi Thu Giang2; Tran Cat Dong3 Ta Manh Hung4; Nguyen Van Bach1 SUMMARY Objectives: To determine and evaluate acyclovir in dogs’ plasma by high performance liquid chromatography spectrometry. Methods: Remove protein in dog plasma by protein precipitation method, then quantify acyclovir by high performance liquid chromatography spectrometry method. Chromatographic conditions were studied; the selection of internal standard; the assessment of the specificity, selectivity, the linear range, lower limit of quantification, extraction efficiency, internal standard and the stability of acyclovir following the guidance of FDA. Results: Acyclovir in dogs’ plasma was performed by liquid-liquid extraction with solvent mixtures diethyl ether-chloroform, felodipin as internal standard; chromatographic conditions including column, mobile phase, a flow-rate, and detector were established. The method had been evaluated and achieved the FDA’s regulations. Conclusion: The high performance liquid chromatography spectrometry method, which would evaluate bioavailability of the acyclovir in experimental dogs, can be used to determine the acyclovir in dogs’ plasma. * Keywords: Acyclovir; Dogs’ plasma; High performance liquid chromatography spectrometry. INTRODUCTION Acyclovir (ACV) is an acyclic guanosine derivative which exhibits a strong and selective inhibition on human viruses including Herpes simplex virus type 1 and 2, varicella-Zoster virus, Epstien-barr virus and cytomegalo. However, ACV has a short half-life, slow and incomplete absorption in the gastrointestinal tract. With the normal oral dose, the amount of absorbed drug is very low (15 - 30%) due to the short stay at the absorption area. As a result, most of dose is excreted through feces (50 - 60%) in the unabsorbed form [1, 2]. In order to overcome the above disadvantages, bio-adherent ACV tablets have been successfully prepared in compliance with the basic standards of Vietnam Pharmacopoeia V and sustained- release bioadhesive tablet formulation. The determination of bioavailability and bioequivalence of the bioadhesive ACV tablets based on the pharmacokinetic parameters is necessary [3]. To assess bioavailability in dogs, we developed and evaluated the high performance liquid chromatography spectrometry (HPLC) method to quantify ACV in dog plasma [4]. 1. Vietnam Military Medical University 2. Hanoi College of Pharmacy 3. Hochiminh City University of Medicine and Pharmacy 4. National Institute of Drug Quality Control Corresponding author: Le Van Thanh (levanthanh09091963@gmail.com) Date received: 04/09/2019 Date accepted: 18/12/2019 Journal of military pharmaco-medicine n o 1-2020 122 MATERIALS AND METHODS 1. Materials and equipment. * Materials and chemicals: - Standard: ACV (potency 99.9%) was provided by National Institute of Drug Quality Control. - ACV bioadhesive tablets (self-prepared): Met basic standards. - Blank dog plasma was suitable for phamaceutical tests. - Other chemicals were of HPLC or analytical grade. * Equipment: HPLC system SHIMADZU; refrigerated centrifuge SARTORIUS (Germany); vortex mixer VELP (Italy); analytical balance (precision 0.1 mg). 2. Methods. * Method development: - Processing of ACV sample in plasma: Plasma samples were removed from the deep freezer and kept in the room temparature to thaw. A volume of 1 mL of plasma was trasferred to a glass tube and 250 µL of percloric acid 20% was added. The resulting solution was votexed for 15 seconds and centrifuged at 10,000 rpm for 15 minutes. The upper organic layer was sucked and injected into the HPLC system. Blank plasma samples with standard ACV and dogs’ plasma after taking the drug were extracted folowing above procedure before being quantified. - Chromatographic conditions: + Chromatographic column: C18; 250 x 4.6 mm, 5 µm. + Column temperature: 400C. + Mobile phase: MeOH:pH 3.0 buffer (8:92) (pH 3.0 buffer: dissolve 0.43 g sodium octansulfonic in 1,000 mL of water, adjust to pH 3.0 with phosphoric acid). + Flow rate: 1.5 mL/min. + Detector: PDA 252 nm. + Injection volume: 100 µL. + Temperature autosampler: 4oC. * Method validation: - Preparation for ACV standard solutions in blank plasma: + Stock solution: Standard stock solution at accurate ACV concentration of about 500 µg/mL in distilled water was prepared. + ACV stock solution was diluted in blank plasma to the concentrations of about 1 µg/mL and 5 µg/mL. + From the above ACV solution, prepare the working standard solutions in blank plasma at accurate ACV concentration from 0.1 - 5 µg/mL. The procedure for evaluating quantitative methods is in accordance with FDA guidelines [5]: - Selectivity: Prepare 6 different dog plasma samples at expected lower quantitative limit (LLOQ) at about 0.1 µg/mL. Analysis of samples by HPLC. - Calibration curve and linear range: Prepare dog plasma samples containing ACV with 7 exact concentrations (0.1; 0.2; 0.5; 1.0; 2.0; 3.0 and 5.0 µg/mL), each concentration included 2 independent samples (with weight adjustment). Analysis of samples by HPLC. Constructing a calibration curve according to linear regression model. - Lower quantitative limit: Prepare 6 dog plasma samples containing ACV at Journal of military pharmaco-medicine n o 1-2020 123 the exact concentration (0.1 µg/mL) (with weight adjustment). Prepare a calibration curve under the same conditions. Analysis of samples by HPLC. Determine the accuracy by comparing the concentration calculated from the calibration curve to the actual concentration. Determine the accuracy by calculating the relative standard deviation RSD. - Inter-day and intra-day precision and accuracy: Prepare 3 batches of dog plasma samples containing ACV, each batch consitsts of 6 independent samples: lower quality control sample (LQC) was about 0.3 µg/mL. Medium quality control sample (MQC) was about 2.5 µg/mL. Higher quality control sample (HQC) was about 4.0 µg/mL. Prepare a calibration curve under the same conditions. Analysis of samples by HPLC. Determine the accuracy of the method by comparing the analytical concentration of the test sample against the actual concentration. Determine the inter-day repeatability by calculating the relative standard deviation of RSD between the analyzed values of each concentration. Intra-day repeatability is determined by repeating the above steps for 3 days. Calculate the relative standard deviation RSD between the analytical value of each concentration on the evaluation dates. - Stability: Prepare 2 batches of dog plasma samples containing ACV, each batch consists of 6 independent samples: LQC sample about 0.3 µg/mL, HQC sample about 4.0 µg/mL. Analyze samples immediately by HPLC method, determine the ACV content in the original sample. After 24 hours, re-assess the samples by HPLC, re-determine the ACV content in the samples above. RESULTS AND DISSCUSION 1. System suitability. Prepare an ACV standard sample in plasma at concentration of about 2.5 ng/mL. Extract it following the procedure. Replicate the injection of this sample for 6 times on HPLC system. Evaluate retention time (TR), peak area and tailing factor in all chromatograms. Table 1: Results of system suitability (n = 6). Sample Peak area (mAU.s) Retention time (s) Tailing factor 1 455080 7.575 1.381 2 451887 7.568 1.383 3 453141 7.567 1.377 4 452448 7.566 1.384 5 450325 7.555 1.384 6 452407 7.557 1.388 Mean 452548 7.565 1.383 RSD (%) 0.3 0.1 0.3 % RSD for the retention time, peak area and tailing factor of ACV standard were less than 2.0%. The obtained value demonstrated the suitability of the system for the pharmaceutical analysis of ACV in biological fluids. 2. Selectivity/specificity. The selectivity/specificity of method was evaluated by comparing the chromatograms obtained from the samples containing ACV standard at LLOQ concentration (0.1 µg/mL) with those obtained from blank samples. Journal of military pharmaco-medicine n o 1-2020 124 Table 2: The interference of blank sample at the retention time (Rt) of ACV. Sample Peak area of blank samples (mAU.s) Peak area of standard samples at LLOQ concentration (mAU.s) 1 2377 19615 2 2782 20542 3 2545 20007 4 1982 19669 5 2289 20808 6 2405 18366 Mean 2397 19835 Peak response ratio (%) 12.1 (a) (b) Figure 1: Chromatogram of blank plasma sample (a) and chromatogram of plasma spiked with ACV standard at LLOQ concentration 0.1 µg/mL (b). The results showed that at the retention time of ACV, there were no peaks in the chromatogram of blank samples, and the peak response of each LLOQ sample was at least 20 times the response of respective blank plasma sample. The analysis method satisfied the requirements of FDA guidelines. Journal of military pharmaco-medicine n o 1-2020 125 3. Calibration curve and LLOQ. Table 3: Correlation between dog plasma ACV concentration and peak area. Sample S1 S2 S3 S4 S5 S6 S7 The nominal concentration (µg/mL) 0.1 0.2 0.5 1.0 2.0 3.0 5.0 Speak (mAU.s) 26639 45027 102556 200375 384079 587020 977053 Regression equation (Y = aX + b)* Y = 193848x + 5048.8 R2 = 0.9999 Concentration determined from the calibration curve (ng/mL) 0.110 0.210 0.503 1.008 1.9553 3.002 5.014 Concentration determined from the calibration curve compared to the nominal value (%) 111.4 103.1 100.6 100.8 97.8 100.1 100.3 Figure 2: The graph shows the correlation between the ACV concentration in dog plasma and the peak area. Re-calculate the concentration of ACV in the standard sample according to the regression equation. The results showed that within the concentration range, 100% of the points achieved the accuracy from the nominal value at the allowable limit (85 - 115%), the R2 value was 0.9999. Thus, within the range of examined concentrations, there was a linear correlation between the concentration of ACV in dogs plasma and the peak area obtained. This linear range was suitable for quantifying ACV in dog plasma. * LLOQ: Analyze ACV samples following the proposed procedure. Determine the back calculated concentration of each LLOQ sample. The accuracy and precision at this concentration were also determined. Concentration (mcg/mL) Journal of military pharmaco-medicine n o 1-2020 126 Table 4: Determination of LLOQ. Samples Peak area of blank plasma samples (mAU.s) Peak area of plasma samples spiked with ACV standard (≈ 0.1 µg/mL) (mAU.s) Back calculated concentration (a) (µg/mL) Accuracy(b) (%) Pass/fail 1 2377 19615 0.091 93.8 Pass 2 2782 20542 0.096 99.0 Pass 3 2545 20007 0.093 96.0 Pass 4 1982 19669 0.091 94.1 Pass 5 2289 20808 0.098 100.5 Pass 6 2405 18366 0.084 86.7 Pass Mean 2397 19835 0.092 95.0 RSD (%) 5.1 5.1 Peak response ratio (LLOQ/BLANK) (%) 8.3 (a: From regression equation; b: % compare with the nominal concentration) The accuracy of each LLOQ sample was higher than 90% and there were not much differences between these samples (RSD was 5.1%), indicating that LLOQ was appropriate. 4. Inter-day and intra-day precision and accuracy. Prepare quality control samples at 3 concentrations: 0.3 µg/mL; 2.5 µg/mL and 4.0 µg/mL (6 samples per concentration). Analyze the samples according to the proposed procedure. Determine the back-calculated concentration of quality control samples and the precision, accuracy at each concentration. Table 5: Intra-day accuracy and precision. LQC (≈ 0.3 µg/mL) MQC (≈ 2.5 µg/mL) HQC (≈ 4.0 µg/mL) Samples Conc.a (µg/mL) Accu. b (%) Conc. a (µg/mL) Accu. b (%) Conc. a (µg/mL) Accu. b (%) 1 0.294 101.1 2.296 94.7 3.802 98.0 2 0.287 98.5 2.448 100.9 3.952 101.8 3 0.291 100.0 2.497 102.9 3.986 102.7 4 0.274 94.3 2.511 103.5 3.984 102.7 5 0.274 94.1 2.513 103.6 4.019 103.6 6 0.280 96.4 2.512 103.6 4.007 103.3 Mean 0.283 97.4 2.463 101.5 3.958 102.0 RSD (%) 3.0 3.0 3.5 3.5 2.0 2.0 (a: From regression equation; b: % compare with the nominal concentration) Journal of military pharmaco-medicine n o 1-2020 127 Table 6: Inter-day accuracy and precision. LQC (≈ 0.3 µg/mL) MQC (≈ 2.5 µg/mL) HQC (≈ 4.0 µg/mL) Day Conc.a (µg/mL) Accu. b (%) Conc. a (µg/mL) Accu.b (%) Conc.a (µg/mL) Accu. b (%) 1 0.294 101.1 2.296 94.7 3.802 98.0 2 0.287 98.5 2.448 100.9 3.952 101.8 3 0.291 100.0 2.497 102.9 3.986 102.7 4 0.274 94.3 2.511 103.5 3.984 102.7 5 0.274 94.1 2.513 103.6 4.019 103.6 I 6 0.280 96.4 2.512 103.6 4.007 103.3 1 0.298 102.3 2.209 91.1 3.733 96.2 2 0.292 100.2 2.453 101.2 3.969 102.3 3 0.288 99.0 2.471 101.9 3.966 102.2 4 0.295 101.4 2.466 101.7 4.003 103.2 5 0.283 97.3 2.440 100.6 3.966 102.2 II 6 0.287 98.5 2.459 101.4 3.970 102.3 1 0.287 96.5 2.498 100.6 4.129 104.0 2 0.291 97.8 2.578 103.9 4.276 107.7 3 0.280 93.8 2.617 105.4 4.250 107.0 4 0.289 97.0 2.620 105.6 4.260 107.3 5 0.283 95.1 2.633 106.1 4.277 107.7 III 6 0.288 96.8 2.644 106.5 4.291 108.0 Mean 0.287 97.8 2.492 101.9 4.047 103.4 RSD (%) 2.3 2.6 4.5 3.8 4.1 3.1 (a: From regression equation; b: % compare with the nominal concentration) The mean accuracy at each quanlify control level was between 85 - 115% and the precision at each quality control level were not exceed 15%. It showed that the analytical method was precise and suitable for analyzing biological fluids. 5. Recovery. Prepare quality control samples in plasma with 5 samples at 3 concentration levels LQC 0.3 µg/mL, MQC 2.5 µg/mL and HQC 4.0 µg/mL. Extract and analyze according to the analytical procedure. Concurrently, analyze samples were prepared in suitable solvent at respective concentrations (without extraction steps). Journal of military pharmaco-medicine n o 1-2020 128 Table 7: Recovery results of ACV. LQC (≈ 0.3 µg/mL) MQC (≈ 2.5 µg/mL) HQC (≈ 4.0 µg/mL) Samples Peak area of ACV in plasma (mAU.s) Peak area of ACV in solvent (mAU.s) Peak area of ACV in plasma (mAU.s) Peak area of ACV in solvent (mAU.s) Peak area of ACV in plasma (mAU.s) Peak area of ACV in solvent (mAU.s) 1 61551 56215 447720 469120 738476 740886 2 60113 55721 477169 464937 767251 740639 3 60913 55531 486505 465630 773939 739433 4 57716 58111 489222 465148 773549 739792 5 57635 57280 489611 463732 780200 740734 6 58890 59172 489519 463607 778021 740505 Mean 59470 57005 479958 465362 768573 740332 RSD (%) 2.8 2.5 3.4 0.4 2.0 0.1 Recovery (%) 104.3 103.1 103.8 The extraction procedure showed a high and stable recovery. RSD % of ACV peak area of samples in solvent (without extraction steps) and in plasma (with extraction steps) at each concentration were ≤ 3.4%. The extent of recovery was consistent with the difference among concentration levels not exceeding ± 1.2%. Therefore, the extraction method and sample procedure preparation were appropriate to apply in experiments. 6. Stability of plasma samples. - Stability of three freeze and thaw cycles: Analysis of LQC and HQC samples according to the established method. Comparison of concentration of ACV in LQC and HQC samples immediately after reconstitution (initial concentration) with storage after 3 freezing - thawing cycles. Table 8: Stability of three freeze and thaw cycles. Concentration (µg/mL) LQC (≈ 0.3 µg/mL) HQC (≈ 4.0 µg/mL) Samples Fresh After three freeze and thaw cycles Fresh After three freeze and thaw cycles 1 0.335 0.310 4.140 4.189 2 0.330 0.311 4.181 4.156 3 0.319 0.312 4.274 4.223 4 0.329 0.320 4.315 4.258 Journal of military pharmaco-medicine n o 1-2020 129 5 0.334 0.315 4.324 4.295 6 0.331 0.320 4.276 4.339 Mean 0.330 0.315 4.251 4.243 RSD (%) 1.7 1.4 1.8 1.6 Variation (%) -4.5 -0.2 After three freeze and thaw cycles, the concentration of ACV remained stable, the difference between the initial concentration and the concentration after 3 cycles was 4.5% (< 15%). RSD % of determination of samples at each concentration was ≤ 1.8% (not exceeding 15%). - Long-term stability of plasma samples: Stability was evaluated at LQC and HQC concentrations. Stored samples at LQC and HQC concentrations at -35ºC ± 5ºC and determined ACV concentration in sample freshly prepared at the first day of long-term stability testing (fresh concentration) and after certain storage time (7.22 and 44 days). Table 9: Long-term stability of plasma samples. Concentration (µg/mL) LQC (≈ 0.3 µg/mL) HQC (≈ 4.0 µg/mL) Samples Fresh After 7 days After 22 days After 44 days Fresh After 7 days After 22 days After 44 days 1 0.335 0.318 0.306 0.299 4.140 4.004 3.997 3.999 2 0.330 0.319 0.304 0.287 4.181 4.091 4.227 4.031 3 0.319 0.348 0.305 0.277 4.274 4.052 4.270 4.127 4 0.329 0.319 0.300 0.285 4.315 4.160 4.351 4.027 5 0.334 0.345 0.305 0.277 4.324 4.181 4.368 4.095 6 0.331 0.338 0.299 0.283 4.276 4.193 4.288 3.955 Mean 0.330 0.331 0.303 0.285 4.251 4.114 4.250 4.039 RSD (%) 1.7 4.3 1.0 2.9 1.8 1.9 3.2 1.6 Variation (%) -0.3 -8.1 -13.7 2.4 0.0 -5.0 Concentration of stored samples (44 days) were ≤ 13.7% (< 15%) compared to fresh concentration. RSD% of determinations of samples at each concentration were ≤ 4.3% (< 15%), demonstrating that the samples remained stable after 44 days of storage to ensure analysis of all samples. Journal of military pharmaco-medicine n o 1-2020 130 CONCLUSIONS Specificity, selectivity, linear range, lower quantitative limit, extraction efficiency and ACV stability in dogs’ plasma were assessed. The process met the requirements of an analytical method in biological fluids according to FDA regulations. This method can be used to quantify ACV in dogs’ plasma when assessing bioavailability and pharmacokinetic parameters of ACV 200 mg bio-adhesive tablets on experimental dogs. REFERENCES 1. Ministry of Health. Vietnam National Pharmacopoeia. Medical Publshing House. 2009, pp.36-40. 2. Sweetman S.C et al. Martindale. Pharmaceutical Press. 2009, pp.862-864, 911. 3. Gaoo J. Bioanalytical method validation for studies on pharmacokinetics, bioavailability and bioequivalence: Highlights of the FDA’s guidance. Asian J Drug Metab Pharmacokinet. 2004, 4 (1), pp.5-13. 4. Center for Drug Evaluation and Research. Guidance for Industry Bioanalytical Method Validation, FDA, U.S. Department of Health and Human Services. 2001. 5. Food and Drug Administration USA. Guidance for Industry: Q1A (R2) Stability Testing of New Drug Substances and Products. 2003.