Bioanalytical Method Development and Validation of Doravirine, Lamavudine and Tenofovir Disoproxil Fumarate using HPLC in Human Plasma
S. Marakatham1*, Dr. P. Shanmugapandiyan2
1Research Scholar, Department of Pharmacy, Prist University Deemed to be University, Thanjavur, Tamilnadu.
2Research Supervisor, Department of Pharmacy, Prist University Deemed to be University,
Thanjavur, Tamilnadu.
*Corresponding Author E-mail: nandhupharm@yahoo.co.in
ABSTRACT:
A novel, simple and sensitive bioanalytical method was developed for estimation of Doravirine, Lamavudine and tenofovir disoproxil fumarate in human plasma with daclatasvir as internal standard. The method was developed using alliance HPLC using Phenomenex C18 (150mm x 4.6mm, 5m) column with mobile phase of 0.01N Potassium dihydrogen phosphate pH (3.5): Acetonitrile (60:40) at flow rate of 1.0ml/min. Detection wavelength was found to be 277nm. The linearity range for doravirine, lamuvidine and Tenfovir was 50-2000ng/ml, 125-5000ng/ml and 20-800ng/ml. Correlation coefficient was 0.999. The method was validated and stability study was carried out as per FDA guidelines.
KEYWORDS: Doravirine, Lamavudine, Tenofovir Disoproxil Fumarate, HPLC, Human plasma.
INTRODUCTION:
With the newly possible aging of the HIV-positive population, patients live longer and receive therapy for several decades; they are exposed to an increased risk of various therapy related and non related adverse effects. This leads to increased pill burden, greater risk of drug-drug interactions, and more drug-related adverse events, which causes compromise in efficacy. This paved the way for single dose treatment.
DORAVIRINE1-5 chemically called as 3 Chloro-5-{1-(4-methyl-5 oxo-4-5-dihydro-1H-1,2,4-triazol-3-yl) methyl}-2-oxo -4-(trifluromethyl)-1-2-dihydro-3-pyridinyl} oxy) benzonitrileIt is a pyridinone non-nucleoside reverse transcriptase inhibitor of HIV-1. This inhibits replication of the virus by non competitive binding, thus conversion of RNA genome to DNA for the proliferation is blocked. Doravirine does not however, inhibit the human cellular DNA polymerases α, ß, and mitochondrial DNA polymerase γ.
LAMUVIDINE: chemically called as 2,3-dideoxy-3-thiacytidine 4-amino-1-[( 2R,5S)-2-(hydroxyl methyl)-1-3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one is a synthetic nucleoside analogue and is phosphorylated intracellularly to its active 5'-triphosphate metabolite, lamivudine triphosphate (L-TP). This nucleoside analogue is incorporated into viral DNA by HIV reverse transcriptase and HBV polymerase, resulting in DNA chain termination.
TENOFOVIR DISOPROXIL is chemically called as bis{[(isopropoxycarbonyl)oxy]methyl} ({[(2R)-1-(6-amino-9H-purin-9-yl)-2-propanyl]oxy}methyl)phosphonate a class of antiretroviral drugs known as nucleotide analogue reverse transcriptase inhibitors . This drug is prescribed in combination with other drugs in the management of HIV infection as well as in Hepatitis B therapy.
LITERATURE REVIEW6-22
According to literature survey there is no bioanalytical method reported for the analysis or estimation of doravirine, lamuvidine and tenofovir disoproxil combination in human plasma. Hence, a novel HPLC method was optimized and validated for simultaneous estimation and validation of doravirine, lamuvidine and tenofovir disoproxil in Human Plasma in synchronization with the USFDA and other relevant Regulatory guidelines
METHODOLOGY:
Materials:
1. API:
Doravirine, Lamivudine and tenofovir API was obtained as a gift sample from Spectrum pharma research solutions..
2. Human plasma :
|
K2 EDTA control plasma |
Deccan Pathological labs, Hyderabad |
Method Development:
Diluent: Based up on the solubility of the drugs, diluent was selected, 0.01NPotassium dihydrogen phosphate and Acetonitrile taken in the ratio of 50:50.
Preparation of Doravirine Stock solution (100µg/ml):
Take 10mg of Doravirine in 100ml volumetric flask and make the volume with diluent to produce 100µg/ml.
Preparation of Doravirine Spiking Solutions:
From the above Doravirine stock solution 0.05ml, 0.1ml, 0.15ml, 0.6ml, 1.0ml, 1.2ml, 1.6ml and 2.0ml was pipette and transferred to 8 individual 10ml volumetric flask and make up the volume up to the mark with diluent to produce 0.5µg/ml, 1.0µg/ml, 1.5µg/ml, 4.0 µg/ml, 10.0µg/ml, 12.0µg/ml, 16.0µg/ml and 20.0 µg/ml.
Calibration standards and quality control (QC) samples were prepared by spiking blank plasma with working stock dilutions of analytes to produce 50ng/ml, 100 ng/ml, 150ng/ml, 400ng/ml, 1000ng/ml, 1200ng/ml, 1600ng/ml and 2000ng/ml.
Preparation of Lamivudine Stock solution (250 µg/ml):
Take 25mg of Lamivudine in 100ml volumetric flask and make the volume with diluent to produce 250µg/ml.
Preparation of Lamivudine Spiking Solutions:
From the above Lamivudine stock solution 0.05ml, 0.1ml, 0.15ml, 0.6ml, 1.0ml, 1.2ml, 1.6ml and 2.0ml was pipette and transferred to 8 individual 10ml volumetric flask and make up the volume up to the mark with diluent to produce 1.25µg/ml, 2.5µg/ml, 3.75µg/ml, 10 µg/ml, 25µg/ml, 30µg/ml, 40µg/ml and 50µg/ml.
Calibration standards and quality control (QC) samples were prepared by spiking blank plasma with working stock dilutions of analytes to produce 125ng/ml, 250 ng/ml, 375ng/ml, 1000ng/ml, 2500ng/ml, 3000ng/ml, 4000ng/ml and 5000ng/ml.
Tenofovir:
Preparation of Tenofovir Stock solution (40 µg/ml):
Take 4mg of Tenofovir in 100ml volumetric flask and make the volume with diluent to produce 40µg/ml.
Preparation of Tenofovir Spiking Solutions:
From the above Tenofovir stock solution 0.05ml, 0.1ml, 0.15ml, 0.6ml, 1.0ml, 1.2ml, 1.6ml and 2.0ml was pipette and transferred to 8 individual 10ml volumetric flask and make up the volume up to the mark with diluent to produce 0.2µg/ml, 0.4µg/ml, 0.6µg/ml, 1.6 µg/ml, 4.0µg/ml, 4.8µg/ml, 6.4µg/ml and 8.0µg/ml.
Calibration standards and quality control (QC) samples were prepared by spiking blank plasma with working stock dilutions of analytes to produce 20ng/ml, 40ng/ml, 60ng/ml, 160ng/ml, 400ng/ml, 480ng/ml, 640ng/ml and 800ng/ml.
Preparation of internal standard Solution (Daclatasvir):
Stock-1: Take 50mg of Daclatasvir in 100ml volumetric flask and make up the volume with diluent to produce 500µg/ml.
Stock-2: From the above solution, take 1ml of solution into 10ml volumetric flask and make up the volume with diluent to produce 50µg/ml solutions.
Final concentration:
From the above solution, take 0.5ml of solution and spiking blank plasma with working stock dilutions of analyte to produce 10µg/ml ISD concentration.
Extraction procedure
Take 750µl of plasma and 500µl of internal standard, 250µl of Doravirine, Tenofovir and Lamivudine from the spiking solutions of both into a centrifuging tube and add 1ml of Acetonitrile go for cyclomixer for 15 sec. Then vertex for 2 min and finally centrifuge for 5 min at 3200rpm speed. After the centrifugation collect the sample and filter it directly inject 10µL into HPLC.750µl of plasma +500µl of internal standard, 250µl of Doravirine, Tenofovir and Lamivudine 15 sec cyclomixer and add 1ml of acetonitrile and do Vertex for 2 min and Centrifuge for 5 min at 3200rpm| and Collection of supernatant sample. Filter the sample (polyvinylidene fluoride or polyvinylidene difluoride 0.45µ filter) and Inject 25µL into HPLC.
RESULTS AND DISCUSSION:
Optimization of Liquid Chromatography:
Chromatographic conditions:
Mobile phase: 0.01N Potassium dihydrogen phosphate pH (3.5): Acetonitrile (60:40)
Flow rate: 1.0ml/min
Column: Phenomenex C18 (150mm x 4.6mm, 5m)
Detector wavelength: 277nm
Column temperature: 300C
Injection volume 20µL
Fig no 1: Optimisation curves of Daclatasvir, Doravirine, Tenofovir and Lamivudine
Observation: All peaks eluted with good peak shape and retention time and tailing was passed.
METHOD VALIDATION:
System suitability of Doravirine, Tenofovir, Lamivudine
Table 1: System Suitability of Doravirine, Tenofovir, Lamivudine
|
Validation No. |
|
SOP No. |
|
Column ID. |
|
|
|
Sample Name |
File Name |
Analyte Area |
Analyte RT (min) |
ISTD Area |
ISTD |
Area |
|
MEAN |
|
2.467 |
|
2.154 |
1.53012 |
|
|
SD |
0.0029 |
0.0077 |
0.016410 |
|||
|
%CV |
0.12 |
0.36 |
1.07 |
|||
|
MEAN |
|
2.467 |
|
2.154 |
0.06281 |
|
|
SD |
0.0029 |
|
0.0077 |
0.000636 |
||
|
%CV |
0.12 |
|
0.36 |
1.01 |
||
|
MEAN |
4.258 |
|
2.154 |
0.36494 |
||
|
SD |
0.0029 |
|
0.0077 |
0.005914 |
||
|
%CV |
0.07 |
|
0.36 |
1.62 |
||
Matrix factor evaluation of Doravirine Tenofovir, Lamivudine
Table no 2: Matrix factor evaluation of Doravirine, Tenofovir, Lamivudine
|
Acquisition Batch ID |
|
Date |
|
|
S. No. |
Plasma Lot No. |
HQC |
LQC |
|
Nominal Concentration (ng/mL) |
|||
|
1600.000 4000.000 640.000 |
150.000 375.000 60.000 |
||
|
(1,360.000-1,840.000) (3,400.000-4,600.000) (544.000-736.000) |
(127.500-172.500) (318.750-431.250) (51.000-69.000) |
||
|
Calculated Concentration (ng/mL) |
|||
|
Mean |
1605.8491 |
151.8221 |
|
|
SD |
14.64089 |
7.03344 |
|
|
% CV |
0.91 |
4.63 |
|
|
% Mean Accuracy |
100.37 |
101.21 |
|
|
Mean |
4008.7555 |
382.3830 |
|
|
SD |
44.24420 |
12.90533 |
|
|
% CV |
1.10 |
3.37 |
|
|
% Mean Accuracy |
100.22 |
101.97 |
|
|
Mean |
652.6277 |
60.3274 |
|
|
SD |
15.35073 |
4.83232 |
|
|
% CV |
2.35 |
8.01 |
|
|
% Mean Accuracy |
101.97 |
100.55 |
|
LINEARITY:
Linearity of Doravirine Tenofovir, Lamivudine
Table no 3: Linearity of Doravirine Tenofovir, Lamivudine
|
S. No |
Final conc. in µg/m |
ISD (area) |
Drug (area) |
Area ratio |
|
1 |
0.05-2 |
599344-603192 |
4580-183213 |
0.008-0.304 |
|
2 |
0.125-5 |
599344-603192 |
10932-437274 |
0.0182-0.7249 |
|
3 |
0.02-0.8 |
599344-603192 |
26436-1050347 |
0.044-1.741 |
Precision and Accuracy Doravirine Tenofovir, Lamivudine
Table no. 4: precision data for intra-day runs of Doravirine Tenofovir, Lamivudine
|
Acquisition Batch ID |
Date |
HQC |
MQC1 |
LQC |
LLOQ QC |
|
Nominal Concentration (ng/mL) |
|||||
|
1600.000 4000.000 640.000 |
1000.000 2500.000 400.000 |
150.000 375.000 60.000 |
50.000 125.000 20.000 |
||
|
Nominal Concentration Range (ng/mL) |
|||||
|
(1,360.000-1,840.000) (3,400.000-4,600.000) (544.000-736.000) |
(850.000-1,150.000) (2,125.000-2,875.0) (340.000-460.000) |
(127.500-172.500) (318.750-431.250) (51.000-69.000) |
(40.000-60.000) (100.00-150.000) (16.000-24.000) |
||
|
Back Calculated Concentration (ng/mL) |
|||||
|
Doravirine |
|||||
|
Mean |
1599.7177 |
998.6880 |
152.1847 |
49.3927 |
|
|
SD |
7.76639 |
9.08521 |
3.31184 |
2.16241 |
|
|
%CV |
0.49 |
0.91 |
2.18 |
4.38 |
|
|
% Mean Accuracy |
99.98 |
99.87 |
101.46 |
98.79 |
|
|
Tenofovir |
|||||
|
Mean |
4000.9167 |
2500.2500 |
375.8833 |
126.6342 |
|
|
SD |
6.42415 |
5.35192 |
3.55825 |
4.91204 |
|
|
%CV |
0.16 |
0.21 |
0.95 |
3.88 |
|
|
% Mean Accuracy |
100.02 |
100.01 |
100.24 |
101.31 |
|
|
Lamivudine |
|||||
|
Mean |
639.9533 |
396.3667 |
61.0667 |
19.6345 |
|
|
SD |
5.80044 |
6.16041 |
3.05347 |
2.16987 |
|
|
%CV |
0.91 |
1.55 |
5.00 |
11.05 |
|
|
% Mean Accuracy |
99.99 |
99.09 |
101.78 |
98.17 |
|
Recovery:
Recovery of Doravirine Tenofovir, Lamivudine
Table no 5: Recovery of Doravirine Tenofovir, Lamivudine
|
Replicate No. |
HQC |
MQC1 |
LQC |
|||
|
Un extracted Response |
Extracted Response |
Un extracted Response |
Extracted Response |
Un extracted Response |
Extracted Response |
|
|
Mean |
185291 61029 352406 |
184838 60384 349116 |
92984 37723 223674 |
91089 37440 218505 |
13840 5684 32888 |
13533 5641 32407 |
|
SD |
1781.39 1015.75 1720.64 |
1309.12 438.57 751.00 |
197.20 335.33 2044.39 |
585.12 331.78 729.73 |
166.24 28.34 429.02 |
250.54 45.22 339.43 |
|
% CV |
0.96 1.66 0.49 |
0.71 0.73 0.22 |
0.21 0.89 0.91 |
0.64 0.89 0.33 |
1.20 0.50 1.30 |
1.85 0.80 1.05 |
|
% Mean Recovery |
99.76 98.94 99.07 |
97.96 99.25 97.69 |
97.79 99.24 98.54 |
|||
|
Overall % Mean Recovery |
98.501 99.145 98.431 |
|||||
|
Overall SD |
1.0899 0.1759 0.6949 |
|||||
|
Overall % CV |
1.11 0.18 0.71 |
|||||
Auto sampler carryover of Doravirine, Tenofovir, Lamivudine
Table no 6: Recovery of Doravirine Tenofovir, Lamivudine
|
Acquisition Batch ID |
|
Date |
|
|
|
Sample ID |
Peak Area |
% Carryover |
||
|
Drug |
ISTD |
Drug |
ISTD |
|
|
Unextracted samples |
||||
|
RS |
0 0 0 |
0 0 0 |
N/A N/A N/A |
N/A N/A N/A |
|
AQ ULOQ |
191923 78734 455497 |
613683 613683 618346 |
0.00 |
0.00 |
|
RS |
0 0 0 |
0 0 0 |
||
|
AQ LLOQ |
4693 1981 11629 |
608237 608237 620190 |
N/A |
N/A |
|
Extracted samples |
||||
|
STD Blk |
0 0 0 |
0 0 0 |
N/A |
N/A |
|
ULOQ |
183213 75957 437274 |
594698 594698 591519 |
0.00 |
0.00 |
|
STD Blk |
0 0 0 |
0 0 0 |
||
|
LLOQ |
4580 1897 10932 |
594553 594553 594666 |
N/A |
N/A |
CONCLUSION:
The validated regulaory bioanalytical method for simultaneous estimation of Doravirine, lamuvidine and tenofovir disoproxil by HPLC in Human Plasma has been developed to support human clinical trials designed to evaluate the safety, pharmacokinetics and efficacy of the compound. The assay was found to be sensitive, selective and reproducible and applied to support the further analysis of doravirine, lamuvidine and tenofovir disoproxil clinical development program.
REFERENCES:
1. https://aidsinfo.nih.gov/drugs
2. https://www.drugbank.ca/drugs
3. https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/210807s000lbl.pdf
4. https://www.ema.europa.eu/en/documents/product-information
5. https://pubchem.ncbi.nlm.nih.gov
6. Anil Dubbaka, Sireesha D, Vasudha Bakshi. Analytical method development and validation for the simultaneous estimation of lamivudine and tenofovir disoproxil fumarate by RP-HPLC method. MOJ Proteomics Bioinform. 2016; 4(5): 306‒309.
7. Madhusudhana reddy Induri1, Bhagavan Raju Mantripragada, Rajendra Prasad Yejella. Development and validation of UPLC method for simultaneous estimation of Efavirenz and Lamivudine in pharmaceutical formulations. Journal of Applied Pharmaceutical Science. 2016; 6 (03): 029-033.
8. G. Bhargavi, Karnaker Reddy. T, Mohammad Younus, Ravindra Reddy. Y. Method development and validation of Lamivudine, Tenofovir and Efavirenz in a combined dosage form by RP-HPLC. Journal of Pharmacy Research. 2012; 5(12): 711-714.
9. Zaheer Ahmed, Sathish Kumar Shetty, Gopinath, Manzoor Ahmed and B. K. Sridhar. Development and validation of RP-HPLC method for the determination of tenofovir disoproxil in bulk and in pharmaceutical formulation. Int. J. Chem. Sci. 2009; 7(4): 2447-2458.
10. Anjaneyulu. N, Nagakishore. R, Nagaganesh. M, Muralikrishna. K, Nithya. A, Sai lathadevi. B, Saikiran Goud. M, Sridevi. N. Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Lamivudine and Tenofovir Disproxil Fumerate in Combined Dosage Form. Asian Journal of Biomedical and Pharmaceutical Sciences. 2013; 3(23): 7-11.
11. MD Abdul Sattar, Suneetha Achanta. Analytical Method Development and Validation for the Determination of Emtricitabine and Tenofovir Disoproxil Fumarate Using Reverse Phase HPLC Method in Bulk and Tablet Dosage Form. J. Pharm. Sci. and Res. 2018; 10(5): 2 1207-1212.
13. Akhilesh Vikram Singh Lila K. Nath Nihar R. Pani. Development and validation of analytical method for the estimation of lamivudine in rabbit plasma. Journal of Pharmaceutical Analysis. 2011; 1(4): 251–257.
14. P. B. Kandagal, D. H. Manjunatha, J. Seetharamappa, S. S. Kalanur. RP HPLC Method for the Determination of Tenofovir in Pharmaceutical Formulations and Spiked Human Plasma. Analytical Letters. 2008; 41: 561–570.
15. Uttam Singh Baghel, Himani Sharma, Anamika Chouhan, Abhay Sharma, Mohammad Mukim, Deeksha Singh. Gradient RP-HPLC Method development for simultaneous estimation of Dextromethorphan hydrobromide, Phenylephrine hydrochloride, and Triprolidine hydrochloride in Liquid Dosage Form. Research Journal of Pharmacy and Technology. 2020;2(13): 583-588.
16. Atish Z. Dhadve, Anupama R. Dashetwar, Dipak P. Kardile, Rajkumar V. Shete. Stability Indicating RP-HPLC Method for Simultaneous Determination of Atorvastain and Nicotinic acid from Tablet Dosage Form. Research Journal of Pharmacy and Technology. 2020; 13(2): 604-608.
17. H.I. Pawar, Lata Kothapalli, Asha Thomas, R.K Nanda and Shivaji Mare. Simultaneous RP-HPLC Method for Estimation of Ezetimibe and Fenofibrate in Synthetic mixture. Research Journal of Pharmacy and Technology. 2008; 1(1): 25-28.
18. R Siva Kumar, MR Santhanakrishnan, P Kumar Nallasivan, R Venkatanarayana. Simultaneous RP-HPLC Method for Estimation of Ezetimibe and Simvastatin in Bulk and Dosage Forms. Research Journal of Pharmacy and Technology. 2008; 1(3): 211-214.
19. Sohan S. Chitlange, Mohammed Imran, Kiran Bagri, DM Sakarkar. A stability-indicating reverse phase high performance liquid chromatography method for the simultaneous determination of ramipril and valsartan in pharmaceutical dosage form. Research Journal of Pharmacy and Technology. 2008; 1(3): 215-217.
20. N Appala Raju, Shabana Begum. Simultaneous RP-HPLC Method for the Estimation of the Emtricitabine, Tenofovir Disoproxil Fumerate and Efavirenz in Tablet Dosage Forms. Research Journal of Pharmacy and Technology. 2008; 1(4): 522-525.
21. Krishna Veni Nagappan, Meyyanathan SN, Rajinikanth B Raja, Suresh Reddy, Jeyaprakash MR, Arunadevi S Birajdar, Suresh Bhojraj. A RP-HPLC Method for Simultaneous Estimation of Ambroxol Hydrochloride and Loratidine in Pharmaceutical Formulation. Research Journal of Pharmacy and Technology. 2008; 1(4): 366-369.
22. Akhilesh Gupta, Swati Rawat, Mayuri Gandhi, Jaydeep Singh Yadav. Method Development and Acid Degradation Study of Doxofylline by RP-HPLC and LC-MS/MS. Asian Journal of Pharmaceutical Analysis. 2011; 1(1): 10-13.
Received on 05.04.2020 Modified on 22.08.2020
Accepted on 07.12.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(8):4087-4091.
DOI: 10.52711/0974-360X.2021.00708