The liquid chromatography–tandem mass spectrometry (LC-MS)/MS methodology was used to develop and validate a method for detecting erythromycin and olaparib in human plasma. Antibiotics such as erythromycin and olaparib fall into this category. Liquid chromatography is used to separate stationary and mobile phases based on differences in their affinities as well as to remove unwanted contaminants. It improves repeatability, sensitivity, resilience, and low-level protein detection. A C18 (C18, 5 m, 100×4.6 mm) column is utilized for high resolution and peak area. The calibration curve is created using linear regression. Internally, telmisartan is utilized as a benchmark. The flow rate of the mobile phase is 0.5 mL/min. Erythromycin and olaparib have mass-to-charge ratios of 735.43–115.97 and 435.08–102.04, respectively. Erythromycin in combination with olaparib resulted in a 98% recovery rate. The precision and accuracy of the results determined by interday QC samples are within acceptable limits. There was no evidence of instability

MADDI, SRINIVAS

N, DEVANNA

RAJAGOPALUDU, PAGIDI

SARITHA, NIMMAKAYALA

English

Innovare Academic Sciences: E-Journals

Vol 15, Issue 4, 2022Online - 2455-3891 Print - 0974-2441METHOD DEVELOPMENT AND VALIDATION OF ERYTHROMYCIN AND OLAPARIB IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRYPAGIDI RAJAGOPALUDU1*, NIMMAKAYALA SARITHA2, DEVANNA N1, SRINIVAS MADDI31Department of Chemistry, Jawaharlal Nehru Technological University, Anantapur, Andhra Pradesh, India. 2Department of Chemistry, Jawaharlal Nehru Technological University College of Engineering, kalikiri, Chittoor, Andhra Pradesh, India. 3AcuBioSys private Limited, Hyderabad, Telangana, India. Email: gopal.gg54@gmail.comReceived: 01 January 2021, Revised and Accepted: 03 March 2022ABSTRACTThe liquid chromatography–tandem mass spectrometry (LC-MS)/MS methodology was used to develop and validate a method for detecting erythromycin and olaparib in human plasma. Antibiotics such as erythromycin and olaparib fall into this category. Liquid chromatography is used to separate stationary and mobile phases based on differences in their affinities as well as to remove unwanted contaminants. It improves repeatability, sensitivity, resilience, and low-level protein detection. A C18 (C18, 5 m, 100×4.6 mm) column is utilized for high resolution and peak area. The calibration curve is created using linear regression. Internally, telmisartan is utilized as a benchmark. The flow rate of the mobile phase is 0.5 mL/min. Erythromycin and olaparib have mass-to-charge ratios of 735.43–115.97 and 435.08–102.04, respectively. Erythromycin in combination with olaparib resulted in a 98% recovery rate. The precision and accuracy of the results determined by interday QC samples are within acceptable limits. There was no evidence of instability.Keywords: Erythromycin, Olaparib, Human plasma, Liquid chromatography–tandem mass spectrometry, Electrospray Ionization.INTRODUCTIONMacrolide antibiotics [1] include erythromycin. Macrolide antibiotics decrease the development of bacteria, which is beneficial for the production of proteins necessary for the survival of bacteria and are used to treat bacterial infections. This includes skin diseases and infections of the respiratory tract. Protein synthesis, which is mediated by ribosomal proteins, is necessary for bacterial reproduction. In vulnerable bacterial organisms, it works as a protein synthesis inhibitor in the 50S ribosome component. It prevents bacterial protein synthesis by preventing the translocation phase in protein synthesis and the gathering of the 50S ribosomal subunit, which leads to the control of various illnesses.Olaparib is an antineoplastic medicine that inhibits the nuclear enzyme Poly ADP Ribose Polymerase (PARP). It is used to treat refractory and advanced ovarian cancer [2]. PARP inhibition may increase the cytotoxicity of DNA-damaging drugs and reverse chemoresistance and radio resistance in tumor cells. The FDA authorized olaparib as the first PARP inhibitor for gBRCAm metastatic breast cancer. The commercial chemotherapeutic medication Lynparza has a generic name of Olaparib. Structure of Erythromycin and Olaparib is shown in (Fig. 1a and 1b).According to the literature survey, several techniques were used for the analysis of erythromycin and olaparib. Erythromycin is analyzed by ultraviolet-spectrophotometry [3], high-performance thin-layer chromatography [4], high-performance layer chromatography (HPLC) [5,6], liquid chromatography–mass spectrometry (LC-MS) [7-10] and olaparib is analyzed by HPLC [11], ultraperformance LC-MS [12], LC-MS [13-15].EXPERIMENTChemicals and reagentsWorking standards for erythromycin and olaparib were obtained from Sigma-Aldrich in Bengaluru, India. For analysis, acetonitrile HPLC grade from Merck, ammonium acetate AR grade from Qualigens fine chemicals, and water HPLC grade from the Milli-Q RO system were utilized for analysis.ChromatographyAn Acquity SM sample manager, an Acquity BSM binary solvent manager, and a thermostated column compartment were included in the system (Waters, Milford, USA). The chromatography was carried out at 40°C on a Phenomenax Luna column (C18, 5 m, 100×4.6 mm). It is set up as 75% A and 25% B for the first 0.5 min, then 10% A and 90% B for the next 3.5 min. Then, it steadily increased to 75% A and 255% B by 3.75 min and stayed there at 5.0 min at a flow rate of 0.5 mL/min.LC-MS/MS conditions and parametersMultiple reaction monitoring was used to perform mass spectrometric detection on a Quattro Micromass quadrapole instrument (Waters, Milford, USA) (MRM). In the turbo electrospray interface, a positive ionization mode was adopted. Table 1 summarizes the functioning parameters of a mass spectrometer.Standard sample preparationTo make a 1.0 mg/mL erythromycin and olaparib solution, pour 100 mg of the working standard solution into a 100 ml volumetric flask and dissolve it with acetonitrile; the final solution is prepared with a 1:1 ratio of water and acetonitrile. The erythromycin and olaparib solutions were obtained and kept in the refrigerator at temperatures below 8°C.RESULTS AND DISCUSSIONValidationIn technique validation, the second stage of the analysis, precise, and accurate data was achieved. Otherwise, it is unfit and inappropriate, according to FDA rules.For concentration determination, calibration curves in the range of 1–1000 ng/mL should be used. Erythromycin and olaparib standards were extracted in the presence of an internal standard and then evaluated using MRM transitions. The analyte to internal standard peak area ratio was computed from the chromatograms. Human plasma was examined without the use of analytes to determine the stability of the sample at room temperature for 24 h.© 2022 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ajpcr.2022v15i4.44044. Journal homepage: https://innovareacademics.in/journals/index.php/ajpcrReview Article29Asian J Pharm Clin Res, Vol 15, Issue 4, 2022, 28-33 Rajagopaludu et al.solution with six different concentration levels was prepared using the same plasma. Intra- and inter-day samples were prepared on alternate days to determine the lowest level. Fig. 2a and 2b shows the calibration curve of erythromycin and Olaparib.MRM transitionsWhen switching from a precursor to a product ion in positive ion mode, multi reaction monitoring is performed. Erythromycin has a mass-to-charge ratio of 435:811, while olaparib has a mass-to-charge ratio of 734:158. Fig. 3a and 3b depict product ion spectra. Table 2 shows the MRM transition conditions summary.LinearityA 1.5 software calibration curve was created by the analyst. The back computed values for erythromycin and olaparib are shown in Tables 3 and 4. The accuracy should be between 85% and 115%. The correlation coefficient for the calibration curve was 0.99.Accuracy and precisionTables 5 and 6 show the intra- and inter-batch precision and accuracy of erythromycin, whereas Tables 7 and 8 show the precision and accuracy of olaparib. The results of the multi-step preparation technique are acceptable, with nominal concentrations ranging from 90% to 110%. The coefficient of variation is <15%, which meets the nominal criterion. Figs. 4 and 5 shows the representative examples of Erythromycin and Olaparib.RecoveryFor erythromycin recovery of LQC area 97.3%, MQC area 99.1% and HQC area is 99.7% and for olaparib LQC area is 96.5%, MQC area 102.7% and HQC area 95.2%. The average recovery of erythromycin is 98.7% and that of olaparib is 98.2%, respectively shown in Tables 9 and 10.StabilityTo acquire LQC, MQC, and HQC values, three cycles of freezing and thawing were performed. For 24 h, the samples are maintained in Table 1: Working parametersCapillary (v) 1.0Cone (v) 30.0Extractor 4RF lens (V) 0Source temperature (°C) 150Desolvation temperature (°C) 500Dwell time per transition 200Cone gas flow (L/hr) 30LM1 resolution 3.6HM1 resolution 14.6Ion energy 1 0.8Entrance 1.0Collision 23Exit 0LM2 resolution 10.5HM2 resolution 14.6Ion energy 2 0.5Construction of calibration curveFive sets of quantitation standards were prepared using the same plasma on alternate days to determine linearity, and a dilute stock Fig. 1: (a) Erythromycin structure, (b) olaparib structurebaFig. 3: (a) Product ion spectra of erythromycin, (b) Product ion mass spectra of olaparib in positive ionization modebaFig. 2: (a) Calibration curve of erythromycin, (b) Calibration curve of olaparibba30Asian J Pharm Clin Res, Vol 15, Issue 4, 2022, 28-33 Rajagopaludu et al.Table 2 : Summary of MRM transition conditionsCompound Mode of ionization Q1 mass (m/z) Q3 mass (m/z) Cone voltage CE Dwell (SEC)Erythromycin Positive 435 281 46 28 0.038Olaparib Positive 734 158 44 44 0.038Table 3: Erythromycin back-calculated standard curve dataConcentration (ng/mL)Std conc. Batch-1 Batch-2 Batch-3 Mean SD % CV % Accuracy0.1 0.08 0.09 0.1 0.09 0.01 11.1 901 0.9 0.82 0.12 0.61 0.43 69.9 61.310 9.6 8.9 10.2 9.57 0.65 6.8 95.650 49.6 48.9 50.1 49.5 0.6 1.2 99.0100 99.7 98.6 99 99.1 0.56 0.5 99.1200 196 189 188 191 4.36 2.2 95.5400 386 392 398 392 6 1.5 98500 489 491 497 492 4.16 0.8 98.41000 990 982 989 987 4.36 0.4 98.7Table 4: Olaparib back-calculated standard curve dataConcentration (ng/mL)Std conc.Batch-1 Batch-2 Batch-3 Mean SD % CV % Accuracy0.1 0.09 0.1 0.08 0.09 0.01 11.11 901 0.9 0.8 0.9 0.87 0.06 6.662 86.6710 9.1 8.9 9.2 9.07 0.15 1.685 90.650 49 47 46 47.3 1.53 3.227 94.6100 96 97 92 95 2.65 2.785 95200 187 191 188 189 2.08 1.103 94.3400 391 389 396 392 3.61 0.92 98500 491 489 497 492 4.16 0.846 98.41000 960 985 989 978 15.7 1.607 97.8Table 5: Erythromycin calculated concentrations obtained for precision and accuracy batchesS. No. Batch-1 Batch-2 Batch-3LQC MQC HQC LQC MQC HQC LQC MQC HQC1 3.6 520 813 4.1 489 810 4.1 496 8202 3.4 517 780 3.8 511 796 3.9 515 7973 3.2 518 794 3.6 521 800 3.8 516 8124 3.1 515 802 4.2 516 803 4.2 513 8105 3 521 791 3.7 521 789 3.6 520 8076 3.9 519 805 3.5 520 812 4.3 518 815MEAN 3.3 518.3 797.5 3.8 513.1 801.6 3.9 513.9 810.1SD 0.3 2.1 11.6 0.2 12.3 8.6 0.2 8.6 7.8%CV 10.0 0.4 1.4 7.3 2.4 1.0 6.6 1.6 0.9LQC: Lower quality control, MQC: Middle quality control, HQC: Higher quality controlFig. 4: (a) Representative example of 0.1 ng/mL chromatogram of erythromycin, (b) representative example of 500 ng/mL chromatogram of erythromycin, (c) representative example of 1 ng/mL chromatogram of Erythromycin, (d) representative example of 50 ng/mL chromatogram for erythromycin, (e) chromatogram 100 ng/mL for erythromycinedcba31Asian J Pharm Clin Res, Vol 15, Issue 4, 2022, 28-33 Rajagopaludu et al.Table 7: Olaparib calculated concentrations obtained for precision and accuracy batchesS. No. Batch-1 Batch-2 Batch-3LQC MQC HQC LQC MQC HQC LQC MQC HQC1 4.1 516 791 3.6 505 823 4.1 510 7972 3.9 518 807 3.8 510 808 3.9 511 8013 3.6 519 789 3.7 509 817 3.6 509 8104 3.7 521 800 3.5 511 799 3.5 505 8165 3.4 517 793 4.1 513 806 3.5 503 8146 3.3 512 796 3.9 515 810 4 512 799Mean 3.6 517 796 3.7 510.5 810.5 3.7 508.3 806.1SD 0.3 3.06 6.6 0.2 3.45 8.4 0.2 3.5 8.1% CV 8.21 0.59 0.8 5.7 0.676 1.0 7.0 0.7 1.0LQC: Lower quality control, MQC: Middle quality control, HQC: Higher quality controlTable 6: Intra- and inter-run precision and accuracy for erythromycin in human plasma Concentration (ng/mL)LQC MQC HQCBATCH-1 (n=6)Intra-run mean 3.37 518.3 797.5Intra-run SD 0.34 2.1 11.6Intra-run % CV 10.1 0.4 1.4Intra-run % Accuracy 84.2 103.6 99.6BATCH-2 (n=6)Intra-run mean 3.82 513.1 801.6Intra-run SD 0.28 12.3 8.6Intra-run % CV 7.3 2.4 1.0Intra-run % Accuracy 95.4 102.6 100.2BATCH-3 (n=6)Intra-run mean 3.98 513 810.1Intra-run SD 0.26 8.6 7.8Intra-run % CV 6.63 1.6 0.9Intra-run % Accuracy 99.6 102.6 101.2INTER-BATCH (n=18)Inter-run mean 3.72 514.7 803.1Inter-run SD 0.39 8.6 10.4Inter-run % CV 10.4 1.6 1.3Inter-run % Accuracy 93.1 102.9 100.3LQC: Lower quality control, MQC: Middle quality control, HQC: Higher quality controlFig. 5: (a) 0.1 ng/mL chromatogram for olaparib, (b) 500 ng/mL chromatogram for olaparib, (c) 1 ng/mL chromatogram for olaparib, (d) 50 ng/mL chromatogram for olaparib, (e) 100 ng/mL chromatogram for olaparibd ecba32Asian J Pharm Clin Res, Vol 15, Issue 4, 2022, 28-33 Rajagopaludu et al.the auto sampler. To achieve bench-top stability, the samples are maintained at room temperature for 4 h. During the entire procedure, no instabilities were discovered. Table 11 shows the stability of analytes.CONCLUSIONSThe current study uses liquid chromatography and mass spectrometry to create and validate erythromycin and olaparib in human plasma. Telmisartan is utilized as an internal standard, and it should be operated in a positive ion mode. Erythromycin has a 98.7% recovery rate, while olaparib has a 98.2% recovery rate. Linearity is measured in ng/mL increments ranging from 0.1 to 1000. The process was found to be stable.CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.Table 9: Erythromycin recovery dataS. No. LQC Area (Counts) MQC Area (Counts) HQC Area (Counts)Aqueous Extracted Aqueous Extracted Aqueous Extracted1 7901 7282 896121 874161 1703411 17121202 8201 7986 877126 862121 1821504 18018913 7634 7531 789143 791722 2123083 19286024 7501 7632 802162 799023 1911202 20231245 7821 7621 823168 807860 2023161 20189266 7932 7704 791260 801251 1900121 1967961MEAN AREA 7831.66 7626 829830 822689 1913747 1908770% RECOVERY 97.3 99.1 99.7AVG. %RECOVERY=98.75, SD=1.229, %CV=1.245, LQC: Lower quality control, MQC: Middle quality control, HQC: Higher quality controlTable 10: Olaparib recovery dataS. No. LQC Area (Counts) MQC Area (Counts) HQC Area (Counts)Aqueous Extracted Aqueous Extracted Aqueous Extracted1 12168 11962 993612 976017 1876321 19245612 10796 9893 891230 890214 1902318 19712063 11384 10321 913216 973205 2134781 19990124 10916 10124 981024 970134 2345671 21093075 9962 10016 892106 901289 2294301 21021756 10614 11284 791260 900631 1967071 1824179Mean Area 10973.333 10600 910408 935248 2086743 1988406% RECOVERY 96.5 102.7 95.2AVG. %RECOVERY=98.2, SD=3.972, %CV=4.044, LQC: Lower quality control, MQC: Middle quality control, HQC: Higher quality controlTable 11: Stability data showing the % CV of analytesAnalyte nameFresh samplesFreeze-thaw stability samplesAuto-sampler stability samplesBench-top stability samplesErythromycinLQC 7.8 3.3 5.6 3.6MQC 5.4 4.6 5.4 7.4HQC 6.5 8.1 6.0 5.3OlaparibLQC 4.8 8.8 6.4 8.3MQC 6.2 5.1 3.2 2.2HQC 7.5 3.9 2.5 4.1LQC: Lower quality control, MQC: Middle quality control, HQC: Higher quality controlTable 8: Intra-and inter-run precision and accuracy for olaparib in human plasmaConcentration (ng/ml)LQC MQC HQCBATCH-1 (n=6)Intra-run mean 3.67 517.1 796Intra-run SD 0.3 3.0 6.6Intra-run % CV 8.21 0.5 0.8Intra-run % Accuracy 91.7 103.4 99.5BATCH-2 (n=6)Intra-run mean 3.77 510.5 810.5Intra-run SD 0.22 3.4 8.4Intra-run % CV 5.74 0.6 1.0Intra-run % Accuracy 94.2 102.1 101.31BATCH-3 (n=6)Intra-run mean 3.77 508.3 806.1Intra-run SD 0.27 3.5 8.1Intra-run % CV 7.06 0.7 1.0Intra-run % Accuracy 94.2 101.6 100.7INTER-BATCH (n=18)Inter-run mean 3.7 512 804.2Inter-run SD 0.2 4.9 9.6Inter-run % CV 6.7 0.9 1.1Inter-run % Accuracy 93.3 102.4 100.5LQC: Lower quality control, MQC: Middle quality control, HQC: Higher quality control33Asian J Pharm Clin Res, Vol 15, Issue 4, 2022, 28-33 Rajagopaludu et al.REFERENCES1. 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METHOD DEVELOPMENT AND VALIDATION OF ERYTHROMYCIN AND OLAPARIB IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY

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METHOD DEVELOPMENT AND VALIDATION OF ERYTHROMYCIN AND OLAPARIB IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY

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