INTRODUCTION:

Efavirenz, (4S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3, 1-benzoxazin-2-one, is a non-nucleoside reverse transcriptase (RT) inhibitor of human immunodeficiency virus type 1 (HIV-1).^[1,2]Efavirenz activity is mediated predominantly by non-competitive inhibition of HIV-1 RT. HIV-2 RT and human cellular DNA polymerases alpha, beta, gamma, and delta are not inhibited by efavirenz.^[3]

The literature survey reveals that there are certain analytical methods available for determination of efavirenz from biological matrices,^[4 ^-9] bulk drug, dosage forms,^[10^-^11] and analytical methods for determination of efavirenz with combination of other antiviral drugs^[12-33] .

Address for correspondence:

M. Gayatri Ramya

Department of Pharmaceutics,

Acharya Nagarjuna University,

Nagarjuna Nagar- 522 510

Received on 07.09.2013 Modified on 15.10.2013

Res. J. Pharm. Dosage Form. & Tech. 6(1): Jan.-Mar. 2014; Page 07-14

Hence the objective of the present research work has been made to develop and validate a simple and stability indicating HPLC method for the analysis of efavirenz which would be highly sensitive, having good resolution and reproducible.

Various validation aspects of the analysis, accuracy, precision, recovery, and the limits of detection and quantification have been measured.

MATERIALS AND METHODS:

Equipment:

The HPLC chromatograph used was Agilent Infinity 1260 series (Agilent Technologies, Santa Clara, CA, USA) equipped with a 1260 binary pump VL (400 bar), 1260 manual injector (600 bar), Rheodyne 7725i seven-port sample injection valve with a 20-μl fixed loop, ZORBAX Eclipse Plus C18 (150 mm × 4.6 mm, 5 μm), UV detector, a standard flow cell (10 mm, 13 μl, and 120 bar; OpenLab CDS EZChrom Ed. Workstation), and 50-μl syringe (FN, LC tip). All weighing for analysis was performed on a Shimadzu electronic analytical balance AX-200 (Kyoto, Japan). Water used for analysis was prepared by triple distillation assembly. All dilutions, mobile phase and other solutions, used for the analysis were filtered through a 0.2-μm nylon filter (Ultipor®N66 Nylon 6,6 membrane, Pall Sciences, Pall India Pvt. Ltd. Mumbai, India).

Preparation of standard solution:

About 100 mg Efavirenz working standard was dissolved in about 10 ml of diluent in a 100 ml volumetric flask. Sonicate to dissolve and then make up to the volume with diluent. Dilute 4ml of the standard stock solution to 100ml with diluent [water: Acetonitrile (1:1)] and mix. Filter through 0.45µm hydrophilic membrane filter.

Preparation of test solution:

Weigh accurately about 50 mg of Efavirenz in a 50 ml volumetric flask, add 5 ml of ACN and then sonicate to dissolve and then make up to the mark with diluent. Dilute 2 ml of the clear solution to 50 ml with diluent. Filter through 0.45 µm Nylon filter.

System suitability:

Chromatograph 10 µl portion of standard preparation into chromatographic system for 5 times. Record the chromatograms and measure the response for the major peak. Tailing factor for Efavirenz peak should not be more than 2.0. Relative standard deviation of Efavirenz peak for 5 replicate injections should not be more than 2%. Inject a 10 µl portion of blank (diluent), standard solution, and test solution into chromatographic system. Record the chromatograms and measure the response for major peak.

RESULTS AND DISCUSSIONS:

Method Validation:

System Suitability:

Inject into chromatograph 10 µl portion of standard solution for 5 times. Record the chromatograms and measure the response for the major peak. Tailing factor for Efavirenz peak should NMT 2.0 of Efavirenz peak for 5 replicate injections should NMT 2%. A typical blank, HPLC chromatogram of standard and sample efavirenz chromatograms were shown in figure 1, 2 and 3 respectively. The system suitability data was also shown in table 1.

Impurity interference:

Prepare impurity solutions in the concentration of 0.15% of standard preparation and inject into the HPLC system. Spike the standard preparation with impurity blend solution at 0.15% concentration and inject into the HPLC system.

Interference from Degradation products:

Acid hydrolysis stress study:

50 mg of Efavirenz was transferred to 50 mL volumetric flask. It was dissolved in diluent and then make up to the mark with diluent.2 mL of the above solution was transferred to a 50 mL volumetric flask. To this 1mL of 2 N hydrochloric acid was added and kept in a water bath at about 60c for 6 hours. After 6 hours it was neutralized with 1mL of 2 N sodium hydroxide solution. Then volume was make up with diluent than Filter through 0.45 µm nylon filter. Chromatograph 10 µl portion of solution into chromatographic system for 2 times. Observe for degradation.

Base hydrolysis stress study:

Weigh the 50 mg of Efavirenz powder and transferred in to 50 mL volumetric flask. It was dissolved in diluent and then make up to the mark with diluent.2 mL of the above solution was transferred to a 50 mL volumetric flask. To this 1mL of 2 N sodium hydroxide solution was added and kept in a water bath at about 60c for 6 hours. After 6 hours it was neutralized with 1mL of 2 N hydrochloric acid. Then volume was make up with diluent than Filter through 0.45 µm nylon filter. Chromatograph 10 µl portion of solution into chromatographic system for 2 times. Observe for degradation.

Peroxide oxidation stress study:

Weigh the 50 mg of efavirenz powder and transferred in to 50 mL volumetric flask. It was dissolved in diluent and then make up to the mark with diluent.2 mL of the above solution was transferred to a 50 mL volumetric flask. To this 1 mL of 5% hydrogen peroxide was added and kept in a water bath at about 60c for 6 hours. After 6 hours the volume was make up with diluent then volume was make up with diluent than Filter through 0.45 µm nylon filter. Chromatograph 10 µl portion of solution into chromatographic system for 2 times. Observe for degradation

Heat stress study:

1 gm of Efavirenz was transferred to a Petri dish, which is kept in a oven maintained at 105c for 6hours. After 6 hours 25 mg of the above sample was transferred to 25 mL volumetric flask. It was dissolved in diluents and then make up to the mark with diluent. 2 mL of the above solution was transferred to a 50 mL volumetric flask, the volume was make up with diluent then volume was make up with diluent than Filter through 0.45 µm nylon filter. Chromatograph 10 µl portion of solution into chromatographic system for 2 times. Observe for degradation.

The detailed information regarding the stress degradation studies were depicted in figures 4 to 7 and the results showed that the drug was susceptible to undergo degradation in all the conditions and the degradation peaks were well resolved from the main drug.

Linearity of test Method:

Six different standard solutions were prepared with concentrations ranging from 80%, 90%, 100%, 110%, 120%. The response for each concentration is determined by assay method. A graph is plotted as concentration vs. peak area. The linearity of the method was shown in table 2.

Precision

Repeatability:

Six different samples were prepared as per the assay test method. Chromatograph 10 µl portion of each solution into chromatographic system for 2 times and calculate RSD assay results.

Accuracy:

To validate whether the assay method can accurately quantify Efavirenz content at various concentrations ranging from 50% to 150% (50%,75%,100%,125%,150%) of the target assay concentration and perform assay in triplicate. The accuracy data of efavirenz was depicted in table 3.

Preparation of 50% spike solution:

Transfer powder equivalent to 25 mg of Efavirenz in to a 50 ml volumetric flask. Add 5 ml of ACN and then sonicate to dissolve and then make up to the mark with diluent. Dilute 2 ml of the clear solution to 50 ml with diluent. Filter through 0.45 µm Nylon filter. Chromatograph 10 µl portion of test solution and blank solution into chromatographic system for 2 times. Observe for interference peaks.

Preparation of 75% spike solution:

Transfer powder equivalent to 37.5 mg of Efavirenz in to a 50 ml volumetric flask. Add 5ml of ACN and then sonicate to dissolve and then make up to the mark with diluent. Dilute 2 ml of the clear solution to 50 ml with diluent. Filter through 0.45 µm Nylon filter. Chromatograph 10 µl portion of test solution and blank solution into chromatographic system for 2 times. Observe for interference peaks.

Preparation of 100% spike solution:

Transfer powder equivalent to 50 mg of Efavirenz in to a 50 ml volumetric flask. Add 5ml of ACN and then sonicate to dissolve and then make up to the mark with diluent. Dilute 2 ml of the clear solution to 50 ml with diluent. Filter through 0.45 µm Nylon filter. Chromatograph 10 µl portion of test solution and blank solution into chromatographic system for 2 times. Observe for interference peaks.

Preparation of 125% spike solution:

Transfer powder equivalent to 62.5 mg of Efavirenz in to a 50 ml volumetric flask. Add 5ml of ACN and then sonicate to dissolve and then make up to the mark with diluent. Dilute 2 ml of the clear solution to 50 ml with diluent. Filter through 0.45 µm Nylon filter. Chromatograph 10 µl portion of test solution and blank solution into chromatographic system for 2 times. Observe for interference peaks.

Preparation of 150% spike solution:

Transfer powder equivalent to 75 mg of Efavirenz in to a 50 ml volumetric flask. Add 5ml of ACN and then sonicate to dissolve and then make up to the mark with diluent. Dilute 2 ml of the clear solution to 50 ml with diluent. Filter through 0.45 µm Nylon filter. Chromatograph 10 µl portion of test solution and blank solution into chromatographic system for 2 times. Observe for interference peaks.

Ruggedness:

System-to-System variability:

To demonstrate the ruggedness of assay method, carry out system to system variability on two HPLC systems (of the same or different manufacture). Standard and test solutions were prepared according to the assay test procedure and the samples were analyzed separately using Agilent and Waters chromatographic systems (MLADL54 & MLADL86).

HPLC Column-to-Column variability:

Standard and test solutions were prepared according to the assay test procedure and the samples were analyzed separately using two different columns by the same manufacture. The results obtained from both the analysis were compared.

Stability of Efavirenz standard and test preparation:

Assay of Efavirenz were performed in duplicate, and the standard and test preparation were kept on bench top for 2 days and analyzed at 1 day intervals up to 2 days against a freshly prepared standard each time.

Stability of Mobile phase:

Assay of Efavirenz were performed in duplicate as per test procedure. By using same lot of mobile phase stored on bench top at initial, analysis was carried after 1 day and 2 days. The results obtained were compared and found to be satisfactory.

Robustness:

Effect of variation in mobile phase composition:

To demonstrate the robustness system suitability parameters were analyzed by injecting standard solution prepared by using two mobile phases, one containing 110% of the method organic phase composition and other contains 90% of the method organic phase composition.

Variation of pH of buffer on mobile phase:

Two mobile phases, one containing the buffer pH 2.8 and other containing the buffer pH 3.2 were prepared. Two assay samples were prepared using different mobile phases and analyzed as per test method. Evaluate the system suitability parameters.

If the Acceptance Criteria fails narrow the organic phase composition range and report the organic phase composition range at which acceptance criteria passes.

Effect of variation in flow rate:

To demonstrate variation in flow rate, the system suitability parameters were checked by injecting standard preparation into the HPLC system with 1.0 ml/min and 1.5 ml/min.

Effect of variation in column temperature:

To demonstrate the robustness of test method, the system suitability parameters were analyzed by injecting standard preparation into the HPLC system at 20C and at 30C. The stability of mobile phase and test preparations were tested and were shown in tables 4 and 5 respectively.

Table 1: System suitability parameter of efavirenz

System suitability	Observation	Acceptance criteria
Tailing factor for Efavirenz peak	1.02	Should be NMT 2.0
RSD for peak area of Efavirenz from 5 injections of standard	0.14	Should be NMT 2.0
Resolution between Efavirenz peak and degradation peak	6.84	Should be NLT 5

Table 2: Linearity of efavirenz

S.NO	% Concentration	Peak area
1	80	976428
2	90	1098641
3	100	1220531
4	110	1342673
5	120	1464627

Figure 1: Blank chromatogram of efavirenz

Figure 2: HPLC chromatogram of standard efavirenz

Figure 3: A Typical HPLC chromatogram of sample efavirenz

Figure 4: A Typical HPLC chromatogram of efavirenz after acid degradation

Figure 5: A Typical HPLC chromatogram of efavirenz after base degradation

Figure 6: HPLC chromatogram of efavirenz after oxidative degradation

Figure 7: HPLC chromatogram of efavirenz after thermal degradation

Table 3: Accuracy of Test Method

Sample No	Spike level	“mg” added	“mg” found	% Recovery	Mean % Recovery
1	50%	25.032	25.102	101.6
2	50%	25.120	25.163	101.1	101.3
3	50%	25.319	25.383	101.3
1	75%	37.478	37.351	99.4
2	75%	37.532	37.598	100.8	100.5
3	75%	37.490	37.493	100.5
1	100%	49.990	50.021	101.5
2	100%	50.100	50.159	100.6	101.0
3	100%	50.190	50.210	100.9
1	125%	67.460	67.450	99.9
2	125%	67.525	67.645	101.4	100.5
3	125%	67.490	67.510	100.3
1	150%	75.120	75.125	100.1
2	150%	74.990	75.123	100.8	100.9
3	150%	75.050	75.100	100.8

Table 4 (a): Stability of Mobile phase

System Suitability Parameters	Observed value			Acceptance Criteria
System Suitability Parameters	Initial day	After1day	After2days	Acceptance Criteria
Tailing factor for Efavirenz peak	1.06	1.00	1.01	NMT 2.0
RSD for peak area of Efavirenz from five injections of standard	0.23	0.12	0.56	NMT 2.0%
Resolution between Efavirenz peak and Uncyclised peak	6.98	6.91	7.23	NLT 5

Table 4 (b): Stability of Mobile phase

Approx. time in days	% Assay	Difference from Initial
Initial	101.1	0.01
1	99.8	1.1
2	100.4	0.7

Table 5 (a): Stability of Efavirenz standard and test preparation

System Suitability Parameters	Observed value			Acceptance Criteria
System Suitability Parameters	Initial day	After 1 day	After 2 days	Acceptance Criteria
Tailing factor for Efavirenz peak	1.01	1.01	1.05	NMT 2.0
RSD for peak area of Efavirenz from five injections of standard	0.23	0.11	0.51	NMT 2.0%
Resolution between Efavirenz peak and Uncyclised peak	6.69	6.75	6.76	NLT 5

Table (5 b): Stability of Efavirenz standard and test preparation

Approx. time in Days	% Assay	Difference from Initial
Initial	100.5	0.02
1	99.1	1.2
2	100.6	0.9

CONCLUSION:

In the present study we developed a new RP-HPLC method for estimation of Efavirenz which is simple, less time consuming using an economical column. The analysis is resolved by using a mobile phase ( Buffer, acetonitrile and triethylamine 30:70:1) at a flow rate of 1.2 ml/min using Symmetry C₁₈, 150 X 4.6mm, 3.5µm. on an isocratic HPLC system. There was no interference observed from other impurities and Efavirenz has been eluted with good peak shape, response within 6 miutes. The assay of the sample has been carried out using this new method and it was found to be 101.0%. The developed method has been validated for different parameters like specificity, accuracy, linearity, precision, ruggedness and robustness. All the results obtained were within the acceptance limits indicating that the developed method is simple, specific, accurate and economical. This method may now be recommended for routine and quality control analysis of efavirenz.

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