Development and Validation of Reverse Phase HPLC Method for Enantiomer Excess Determination of Tenofovir Disoproxil Fumarate drug Substance

 

Ch. Ramesh^1,2*, D. Rama Devi³ , MNB. Srinivas^1,2, Nagaraju Rajana¹, S. Radha Krishna²,

K. Basavaiah¹*

¹Department of Inorganic and Analytical Chemistry, Andhra University, Visakhapatnam - 530003, India.

²Laurus Labs Ltd., Visakhapatnam-531021, India.

³A.U. College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, 530003, India.

 

ABSTRACT:

A stereospecific RP-HPLC method for the separation and estimation of S-isomer content in tenofovir disoproxil fumarate drug substance was developed and validated on a reverse-phase amylose derivativesed chiral column. The effect of organic modifiers, namely, methanol, acetonitrile and triethylamine in mobile phase was optimized as 0.1% triethyl amine in mixture of water, methanol and acetonitrile (10:75:15, v/v/v) to obtain the best enantiomeric separation. UV detection was performed at 260nm. The flow rate was kept at 0.8 mL/min and the column temperature was set at 25°C. The retention times of tenofovir and its S-isomer were observed to be 5.137 min and 8.768 min respectively. The linear regression analysis data for calibration plots showed a good linear relationship over a concentration range of 0.0005mg/mL – 0.01mg/mL for S-isomer. The values of the correlation coefficient were 0.999 for S-isomer. The limit of detection (LOD) was 0.0001 mg/mL and the limit of quantification (LOQ) was 0.0005mg/mL. The precision of S-isomer at LOQ level was evaluated through six replicate injections and the % RSD of the peak response achieved is 3.07. The percentage recoveries of S-isomer from tenofovir disoproxil fumarate were ranged from 96.4% to 102.2%. The proposed method was found to be accurate, precise and suitable for the separation and enantiomer excess determination of unwanted S-isomer in active pharmaceutical ingredients (API). The analytical results were supported by statistical parameters. The proposed method could be successfully applied to the enantiomeric purity analysis of tenofovir disoproxil fumarate in drug substance. This method was validated in as per ICH Q2 (R1) and USP validation of compendial methods <1225>.

 

 

 

 

INTRODUCTION:

Tenofovir disoproxil fumarate (TDF) is chemically it is [[(2R)-1-(6-aminopurin-9-yl) propan-2-yl]oxymethyl-(propan-2-yloxycarbonyloxymethoxy) phosphoryl] oxymethyl propan-2-yl carbonate;(E)-but-2-enedioic acid and it’s molecular formula is C₁₉H₃₀N₅O₁₀P, C₄H₄O₄ with molecular weight 635.5 g/mol. The drug molecule contains one chiral center and the configuration of the molecule is “R” (figure-1).^1,2 TDF is the first nucleotide analogue approved for HIV-1 treatment and it is used in combination with other antiretroviral for the treatment of HIV infections. TDF belongs to the class of antiretroviral drugs known as nucleotide analogue reverse transcriptase inhibitors (nRTIs), which blocks reverse transcriptase, an enzyme crucial to viral production in HIV infected people.^3-7

 

Chiral compounds often exhibit considerably different biological activities due to the highly specific nature of the chiral ligand-recognition site interaction. The enantiomers, along with the corresponding racemates, require specific biological, pharmacological and toxicological studies before being launched in clinical practice.^8-10 The US Food and Drugs Administration (FDA) and the European Committee for Proprietary Medicinal Products have required manufacturers to research and characterize each enantiomer in all drugs proposed to be marketed as a mixture. Therefore, racemic drugs ceased to be a rational commercial option, and academia and drug firms have been developing synthetic procedures and chiral separation techniques to achieve enantiomerically pure biologically active compounds.^11,12 Chromatography on solid stationary phases is one of the most powerful separation techniques as wide spectrum of possibilities offered by adjusting both the liquid mobile phase and the solid stationary phase to achieve the separation.^13,14

 

Few analytical methods were reported in literature for determination tenofovir disoproxil fumarate in drug substance and also for estimation of S-isomer by normal phase chiral HPLC.^15-19

 

Author has been developed and validated reverse phase chiral HPLC method with the objective of high resolution between the S and R isomers of tenofovir disoproxil fumarate, lower limit of quantification (LOQ), lower limit of detection (LOD) and higher degree of reproducibility along with very short run time.

 

Figure 1: Chemical structure of (a) Tenofovir disoproxil fumarate and (b) S-isomer

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Active pharmaceutical ingredient of tenofovir disoproxil fumarate and Tenofovir s-isomer were synthesized by process research development of Laurus Labs Limited. The triethylamine (GR grade), methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from Rankem, India. High purity water was prepared using a millipore reference A⁺ (Millipore, Milford, MA, USA) purification system.

 

Instrumentation:

HPLC system with make Shimadzu LC-2010 CHT and weights measurements are taken by using analytical balance with make Mettler Toledo XS205 Dual Range. Data was processed through LC solution software.

 

Chromatographic conditions:

A column used is Chiral pak AD-H (250 × 4.6mm, 5μm) as stationary phase with a mobile phase 0.1% triethylamine in mixture of water, methanol and acetonitrile (10:75:15, v/v/v) at a flow rate of 0.8mL min^-1 and UV detection wave length at 260nm and 20µL sample was injected for 15 minutes.

 

Preparation of mobile phase:

Mobile phase:

Mixed 100mL of water, 750mL of methanol and 150 mL of acetonitrile. To this mixture, added 1.0mL of triethylamine and mixed well, filtered through 0.45µm filter paper and degassed.

 

Diluent:

Mixed 500mL of water and 500 mL of methanol, filtered through 0.45µm filter paper and degassed.

 

Preparation of system suitability solution:

Weighed 12.5mg of tenofovir disoproxil fumarate and 12.5mg of Tenofovir S-isomer standard and transferred into 25mL volumetric flask, dissolved and diluted to volume with diluent and mixed well.

 

Preparation of standard solution:

Weighed 25mg of tenofovir disoproxil fumarate standard and transferred into 25mL volumetric flask, dissolved and diluted to volume with diluent and mixed well.

 

Preparation of sample solution:

Weighed 25mg of tenofovir disoproxil fumarate sample and transferred into 25mL volumetric flask, dissolved and diluted to volume with diluent and mixed well.

 

RESULTS AND DISCUSSION:

Method development and method optimization:

Two different amylose and cellulose based stationary phase columns, Chiralpak AD-H (amylose tris-3,5-dimethylphenylcarbamate) and Chiralcel OD-H (cellulose tris-3,5-dimethylphenylcarbamate) were evaluated for method development activity, using methanol and acetonitrile as organic modifiers in water. The chiral recognition mechanism on these chiral stationary phases (CSPs) is generally due to the formation of solute-CSP complexes through inclusion of enantiomers into the chiral cavities in the higher order structures of the CSPs. The amylose and cellulose-based CSPs belongs to a class of helical polymers possessing a one-handed helical conformation after proper modification of the reactive hydroxyl groups into esters and carbamate. The carbamate groups can interact with solutes through hydrogen bonding using C=O and N-H groups, and through dipole dipole interaction using C=O moiety. Tenofovir disoproxil fumarate contains a functional N–H group and can form hydrogen bonds with C=O group in the CSPs.^20-22 A chiralcel OD-H column did not show selectivity for tenofovir disoproxil fumarate enantiomers with methanol and acetonitrile as organic modifiers in water. However, little selectivity (resolution of 1.26) was shown while using a Chiralpak AD-H column with 25 % acetonitrile in methanol and water. A higher resolution was obtained with 15% acetonitrile. Triethylamine was added (0.1%) to the mobile phase for better peak shapes. Finally the method was optimized using a mobile phase containing 0.1 % triethylamine in mixture of water, methanol and acetonitrile (10:75:15, v/v/v). Column temperature was maintained at 25°C. This method was validated in as per ICH Q2 (R1) and USP validation of compendial methods <1225>.^{23, 24}

 

Optimization of chromatographic conditions:

A rugged and suitable reverse phase HPLC method for the separation of the enantiomers, stationary phases and mobile phase were employed. Chiralpak AD-H (250 x 4.6)mm, 5µm with mobile phase consisting of 0.1% triethylamine in mixture of water, methanol and acetonitrile (10:75:15, v/v/v). The flow rate was kept at 0.8mL/minute and the detection wavelength was set at 260 nm and the column temperature was set at 25^˚C. The proposed method was productively applied for the quantitative determination of S-isomer in tenofovir disoproxil fumarate drug substances form.

 

The linear regression analysis data for calibration plots showed a good linear relationship over a concentration range of 0.0005mg/mL–0.01mg/mL for S-isomer. The mean values of the correlation coefficient were 0.999 for S-isomer. The method was validated as per ICH guidelines. The limit of detection (LOD) was 0.0001 mg/mL and the limit of quantification (LOQ) was 0.0005 mg/mL. The precision of S-isomer at LOQ level was evaluated through six replicate injections and the % RSD of the peak response was achieved as 3.07%.

 

The percentage recovery of S-isomer from tenofovir disoproxil fumarate was ranged from 96.4% to 102.2%. The developed and validated HPLC method and the statistical analysis showed that the method is repeatable and selective for the estimation of the S-isomer in tenofovir disoproxil fumarate. The optimized chromatographic conditions were shown in the table-1.

 

Table 1: Chromatographic conditions

Column	Chiral pak AD-H Column (250x 4.6) mm, 5µm
Wavelength	260 nm
Flow rate	0.8 mL/min
Column oven temperature	25°C
Mobile phase	0.1% Triethyl amine in mixture of water, methanol and acetonitrile (10:75:15, v/v/v)
Sample temperature	25 °C
Run time	15 minutes

 

Method validation:

Specificity:

The specificity of the method is performed by injecting tenofovir disoproxil fumarate and S-isomer individually and mixture of both tenofovir disoproxil fumarate and S-isomer. The specificity determined by using resolution between tenofovir disoproxil fumarate and S-isomer. Baseline separation with a resolution of more than 5.7 achieved and results were shown in table-2. The blank, tenofovir disoproxil fumarate, S-isomer standards and System suitability chromatogram of tenofovir disoproxil fumarate were shown in figure-2, 3 and 4.

 

Figure 2: Blank chromatogram of tenofovir disoproxil fumarate

 

Figure 3: Standard chromatograms of Tenofovir disoproxil fumarate and S-isomer

 

Figure 4: System suitability chromatogram of Tenofovir disoproxil fumarate

 

 

Table 2: System suitability results

Name	Retention time (minutes)	Plate count	Tailing factor	Resolution
Tenofovir disoproxil fumarate	5.137	3255	1.25	---
S-isomer	8.768	1520	1.15	5.76

 

Limit of Detection (LOD) and Quantitation (LOQ):

The LOD and LOQ for tenofovir disoproxil fumarate (R-isomer) and S-isomer were determined at a signal to noise ratio of 3:1 and 10:1 respectively, by injecting a series of diluted solution with known concentration of tenofovir disoproxil fumarate and S-isomer. Precision study was carried out at the LOQ level by injecting six replicate injections individually and the %RSD for the area counts was calculated. The limit of detection, limit of quantification and precision at LOQ for tenofovir disoproxil fumarate and S-isomer are reported in table-3.

 

Precision:

Method reproducibility was determined by measuring repeatability and intermediate precision of retention times and peak areas for each enantiomer. The repeatability of the method was determined by analyzing six replicate injections containing S-isomer (0.005 mg/mL).

 

The study was performed on different day, using different column and instrument and the same protocol was followed for two different days to study inter-day variation, prepared different solutions on different days. The %RSD of S-isomer found in the spiked samples was calculated.

 

The RSD (%) of peak area for S-isomer in the study of the repeatability was shown in table-3. Results for intermediate precision (intra and inter day repeatability) are within 2.0%. These results confirmed that the method is highly precise.

 

Accuracy:

The accuracy of the method was carried out by injecting a known concentration of S-isomer to the tenofovir disoproxil fumarate drug substance. The accuracy was calculated in terms of recovery (%) and the obtained chromatogram was shown in figure-5. It is observed that the percentage recovery of S-isomer in drug substance samples ranged from 96.8 % to 102.2 % and results are reported in table-4.

 

Linearity:

Detector response linearity was assessed by preparing calibration sample solutions of S-isomer covering from 0.0005mg/mL to 0.01mg/mL in diluent. The regression curve was obtained by plotting area versus concentrations, using the least square method. The correlation coefficient, slope and Y-intercept of the calibration curve was calculated and the obtained plots are shown in figure-6.

 

For S-isomer, linear calibration curve was obtained ranging from LOQ, 20 %, 50 %, 80 %, 100 %, 120 %, 150 % and 200 %. The correlation coefficient obtained is greater than 0.999. The results indicate excellent linearity and shown in table-4.

 

Table 3: LOD, LOQ, Precision at LOQ and Precision at specification level

Solution	Concentration (mg/mL)	Tenofovir disoproxil fumarate (Area)	S-isomer (Area)
LOD	0.0001	1982	3085
LOQ	0.0005	12817	13735
		12658	12841
		12419	13453
		12341	12658
		12754	13001
		12549	12985
	Average	12589.6	13112.1
	STDEV	187.6	403.0
	%RSD	1.49	3.07
Specification level	0.005	-	154273
		-	153137
		-	155180
		-	156224
		-	153100
		-	155798
	Average	-	154618.6
	STDEV	-	1334.4
	%RSD	-	0.86

Figure-5: Zoomed Chromatogram of Tenofovir disoproxil fumarate and S-isomer at 0.5% level.

 

Table 4: Accuracy and Linearity of S-isomer

Accuracy			Linearity of S-isomer
S. No.	% Area of S-isomer	% Recovery	Level	Concentration (mg/mL)	Area of S- isomer
1	0.484	96.8	LOQ	0.0005	13735
2	0.482	96.4	20	0.0010	29180
3	0.511	102.2	50	0.0025	76791
4	0.495	99.0	80	0.0040	120464
5	0.502	100.4	100	0.0050	153137
6	0.487	97.4	120	0.0060	186458
			150	0.0075	234516
			200	0.01	308742
			Regression coefficient		0.9998
			Slope		31215987
			% Y-intercept		-2045

 

Figure 6: Linearity plot for S-isomer

 

Analytical Solution Stability:

Solution stability was studied by keeping the test solution in tightly capped volumetric flask at room temperature on a laboratory bench for 48 hr. Content of S-isomer was checked for every 12 hr interval and compared with freshly prepared solution. No variation was observed in the content of S-isomer for the study period and this indicates tenofovir disoproxil fumarate sample solutions prepared in diluent were stable up to 48 hr at room temperature.

 

Mobile phase stability was carried out by evaluating the content of S-isomer in tenofovir disoproxil fumarate sample solutions, which were prepared freshly at every 12 hr interval for 48 hr. The same mobile phase was used during the study period. No variation was observed in the content of S-isomer for the study period and it indicates prepared mobile phase was found to be stable up to 48 hr at room temperature.

 

Robustness:

To determine the robustness of the method, flow rate was changed from 0.7 to 0.9 mL/min. The effect of a change in the percent acetonitrile, column temperature at 20° C and 30° C instead of 25 °C were studied, and the other chromatographic conditions were held constant stated previously and obtained results of robustness is in table-5.

Table 5: Robustness data

Parameter altered	Resolution
Flow rate
0.7 mL/min	5.82
0.8 mL/min	5.76
0.9 mL/min	5.41
Column temperature
20° C	5.79
25° C	5.76
30° C	5.55
Acetonitrile ratio
10	6.21
15	5.76
20	4.13

 

Table 6: Method validation parameters

Validation Parameter	Criteria	Result
Specificity	Resolution	5.76
LOD (mg/mL)	(R)-Enantiomer (mg/mL)	0.0001
LOQ (mg/mL)	(R)-Enantiomer (mg/mL)	0.0005
System precision (n = 6)	% RSD for area	0.89
Method precision (n = 6)	% RSD for area	1.22
LOQ precision (n = 6)	% RSD for area	3.07
Accuracy	% Recovery	96.4 to 102.2
Linearity	Correlation coefficient	0.9998
	Slope	31215987
	% Y-intercept	-2045

 

CONCLUSION:

A simple, specific, linear, accurate and precise reverse phase chiral HPLC method was successfully developed, which was capable of separating the undesired enantiomer from tenofovir disoproxil fumarate, column Chiralpak AD-H (250 mm X 4.6 mm, 5.0µm) was found to be selective for enantiomers of tenofovir disoproxil fumarate. The developed and validated method can be used for the estimation of the S-isomer in tenofovir disoproxil fumarate drug substance.

 

ACKNOWLEDGEMENTS:

The author thanks the Management of Laurus Labs Ltd., Visakhapatnam, India for permitting this work to be published.

 

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interests regarding the publication of this article.

 

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Received on 09.06.2020                    Modified on 04.07.2020

Accepted on 23.07.2020                   ©AJRC All right reserved