Development and Validation of a Simple, Sensitive, Selective and ...

29 downloads 60488 Views 1MB Size Report
Sep 3, 2014 - The active pharmaceutical ingredient (API) and standards of im- purities were ... For Permissions, please email: [email protected]. Journal of .... erence standard, and the mass balance (% assay + % impurities +.
Journal of Chromatographic Science 2015;53:662– 675 doi:10.1093/chromsci/bmu097 Advance Access publication September 3, 2014

Article

Development and Validation of a Simple, Sensitive, Selective and Stability-Indicating RP-UPLC Method for the Quantitative Determination of Ritonavir and Its Related Compounds Srinivasarao Koppala1,2*, Bibhuranjan Panigrahi1, S.V.N. Raju1, K. Padmaja Reddy1, V. Ranga Reddy1 and Jaya Shree Anireddy2 1

Analytical Research and Process Development, Integrated Product Development Operations, Dr Reddy’s Laboratories Ltd, Bachupally, Qutubullapur, Ranga Reddy District 500 072, Andhra Pradesh, India, and 2Centre for Chemical Sciences and Technology, IST, J. N. T. University, Kukatpally, Hyderabad 500085, Andhra Pradesh, India *Author to whom correspondence should be addressed. Email: [email protected], [email protected] Received 4 January 2014; revised 19 June 2014

A simple, sensitive, selective and reproducible stability-indicating ultra-performance liquid chromatographic method was developed for the quantitative determination of degradation products and process-related impurities of Ritonavir in a pharmaceutical dosage form. Chromatographic separation was achieved on a polar embedded Waters Acquity BEH Shield RP18 (100 3 2.1 mm, 1.7 mm) column thermostated at 5088 C under gradient elution by using a binary mixture of potassium dihydrogen phosphate (0.01 M, pH 3.5) and acetonitrile at a flow rate of 0.5 mL/min. Chromatogram was monitored at 240 nm using a photodiode array detector. The drug and its related impurities are eluted within 20 min. To prove the stability-indicating power of the method, the drug was subjected to hydrolytic (acid, alkaline and water), oxidative, photolytic and thermal stress conditions. The unknown degradants were identified by the LC–MS-MS method, which revealed protonated molecular ion peaks [M 1 H]1 at m/z 551.40 for hydrolytic degradants, and m/z 737.60 and m/z 753.40 for photolytic degradants. A plausible mechanism for the formation of degradation and process impurities was proposed. The performance of the method was validated according to the International Conference on Harmonization guidelines.

the stability-indicating HPLC method as per the European Pharmacopoeia and United State Pharmacopoeia (USP) (20, 21). In these methods, a YMC-Pack C4 column with quaternary mixture of potassium dihydrogen phosphate (0.03 M), acetonitrile (ACN), tetrahydrofuran and n-butanol were used as the mobile phase. The mobile phase flow was 1.0 mL/min in gradient elution mode at a column temperature of 608C. The reported method has longer run time (155 min) and incapable of estimating the major degradants and potential impurities, i.e., Imp-1, Imp-2, Imp-5, Imp-7 and Imp-10. In contrast, the present communication comprising of the UPLC method is capable of estimating RIT and all its process and degradation impurities with a shorter run time (20 min) using the simple and cost-effective mobile phase. The literature survey reveals that no reference exists for the quantitative determination of impurities by a stability-indicating UPLC method. Hence, it was felt necessary to develop an accurate, rapid, selective and sensitive stability-indicating LC method for the determination of RIT and its related compounds. This method was successfully validated according to the International Conference on Harmonization (ICH) guidelines (22). Experimental

Introduction Ritonavir (RIT) is chemically known as 5-thiazolylmethyl[(aS)a-[(1S,3S)-1-hydroxy-3-[(2S)-2-[3-[(2-isopropyl-4-thiazolyl) methyl]-3-methylureido]-3-methylbutyramido]-4-phenylbutyl] phenethyl]carbamate (Figure 1A). The drug is approved by United States Food and Drug Administration and marketed under the trade name of NORVIR (RIT tablets, capsules and oral solution formulation) by Abbott Laboratories. RIT is a peptidomimetic inhibitor of both the HIV-1 and HIV-2 proteases. It is widely used in the treatment against the acquired immune deficiency syndrome and prescribed in combination with other antiretroviral drugs (http://www.accessdata.fda.gov/drugsatfda_ docs/label/2008/020945s022,020659s042lbl.pdf; accessed June 1, 2013). An extensive literature search revealed only a few analytical techniques for determining RIT, including the following methods: LC – MS estimation (1), HPLC estimation (2 – 15), UV spectrophotometry (16, 17) and high-performance thin-layer chromatography (18, 19). All of the listed techniques are applied to the assay of RIT. According to our findings, none of the currently available analytical methods is stability indicating with respect to quantification of all known and degradation impurities, with the exception of

Materials and reagents The active pharmaceutical ingredient (API) and standards of impurities were supplied by Dr Reddy’s Laboratories Limited, IPDO, Hyderabad, India. Commercially available NORVIR tablets were used as dosage form for analysis. The HPLC grade ACN and analytical grade potassium dihydrogen phosphate (KH2PO4), ortho phosphoric acid (H3PO4), hydrochloric acid (HCl), sodium hydroxide (NaOH) and hydrogen peroxide (H2O2) were purchased from Merck, Darmstadt, Germany. Deionized water was prepared using a Milli-Q plus water purification system from Millipore (Bedford, MA, USA). Instrumentation The system used for method development, forced degradation and validation study was Waters Acquity UPLC system equipped with a 2996 photodiode array (PDA) detector. The output signal was monitored and processed using Empower 2 software (Waters Corporation, Milford, MA, USA). Chromatographic conditions Separation was accomplished on a Acquity UPLC BEH Shield RP-18 column (100 mm, 2.1 mm and 1.7 mm). The mobile

# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Figure 1. Chemical structure and name of RIT and impurities.

phase A contains a mixture of 0.01 M KH2PO4 buffer ( pH 3.5 adjusted with H3PO4) and ACN in the ratio of 80 : 20 (v/v). The mobile phase B contains a mixture of Milli-Q water and ACN in the ratio of 20 : 80 (v/v). The mobile phases were filtered through a

nylon 0.2-mm membrane filter. The flow rate of mobile phase was 0.5 mL/min. The UPLC gradient program (time in min/%B) was set as 0.01/17, 0.6/17, 6/30, 11/45, 18/75, 18.01/17 and 20/17. The column temperature was maintained at 508C. The detection Quantitative Determination of RIT and Its Related Compounds 663

Figure 1. Continued

was monitored at a wavelength of 240 nm. The injection volume was set as 3.0 mL. A mixture of buffer (0.01 M KH2PO4, pH 3.5) and ACN in the proportion of 50 : 50 (v/v) was used as a diluent.

LC –MS-MS conditions LC – MS-MS system (Agilent 1200 series liquid chromatography coupled with Applied Biosystems 4000 QTrap triple quadrupole mass spectrometer with Analyst 1.4 software, MDS SCIEX, USA) was used for identification of the unknown compounds formed during forced degradation studies. Hypersil BDS C18 (250  4.6 mm, 5 mm column) was used as stationary phase. Ammonium acetate (0.01 M; pH 3.5 adjusted with formic acid) was used as buffer. Buffer and ACN in the ratio of 80 : 20 (v/v) were used as mobile phase A. Water and ACN in the ratio of 20 : 80 (v/v) were used as mobile phase B. The gradient program (time in min/% B) was set as 0.01/20, 5/20, 45/80, 50/90, 50.1/20 and 55/20. Buffer and ACN in the ratio of 1 : 1(v/v) were used as 664 Koppala et al.

diluent. The flow rate was 1.0 mL/min with an injection volume of 10 mL. The analysis was performed in positive electrospray ionization mode. The ion source voltage was 5,000 V. The source temperature was 4508C. GS1 and GS2 are optimized to 30 and 35 psi, respectively. The curtain gas flow was 20 psi. For fragmentation (MS/MS) studies, collision energy and declustering potential were set at 30 eV and 30 V, respectively.

Preparation of stock and standard solutions A stock solution of RIT (3,000 mg/mL) was prepared by dissolving the drug in diluent. Working standard solutions of 1.5 and 300 mg/mL were prepared from the stock solution for the determinations of the related compounds and assay, respectively. The individual stock solutions (75 mg/mL) of all impurities were prepared in the diluent. These solutions were prepared freshly and diluted further quantitatively to study the validation attributes. The specification limits considered for validation studies were

0.15% for Imp-1, Imp-2, Imp-5, Imp-7 and Imp-10; 0.1% for Imp-3, Imp-4, Imp-6, Imp-8, Imp-9, Imp-11 and Imp-13 and 0.2% for Imp-12. Impurities blend solution was prepared by dissolving RIT (1,500 mg/mL) and all impurities in diluent at the specification level. This impurities blend solution was used for method development.

Preparation of sample solution NORVIR 100 mg tablets (n ¼ 20) were weighed and averaged before being crushed into fine powder. Powder equivalent of 150 mg RIT was dissolved in diluent with shaking for 10 min and sonication for 10 min to give a solution containing 1,500 mg/mL. This solution was filtered through a 0.45-mm nylon membrane filter and used for related substances. The solution was further diluted to 300 mg/mL (10.0 – 50 mL with diluent) and used for assay. Similarly, API test preparation was prepared by dissolving 15 mg of RIT to 10 mL with diluent.

Specificity and mass balance study Stress degradation studies were performed according to ICH guidelines Q1A (R2) (23) to demonstrate the stability-indicating nature and specificity of the proposed method. About 75 mg, each of RIT drug substance was transferred into four separate 50 mL volumetric flasks and subjected to forced degradation study under acid (1 M HCl at 708C for 5 h), base (0.001 M NaOH at room temperature for 30 min), neutral (water at 708C for 5 h) and oxidation (5.0%, v/v, H2O2 at room temperature for 48 h). The stressed samples of acid and base degradation were neutralized with 1 M NaOH and 0.001 M HCl, respectively, and made up to volume with the diluent. The drug was placed in a thermally controlled oven at 908C up to 7 days for thermal stress study. Photolytic degradation was performed by exposing the drug to visible light and UV with minimum exposure of 1.2 million lux-hours and 200 W h/m2, respectively. A peak purity test was carried out for the RIT peak by using a PDA detector in all stressed samples. An assay of stressed samples was performed (at 300 mg/mL) by comparison with qualified reference standard, and the mass balance (% assay þ % impurities þ % degradation products) was calculated. The assay was also calculated for the RIT sample by spiking all 13 impurities at the specification level.

Validation parameters The proposed method was validated according to the ICH guidelines for its limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy, solution stability and robustness. Moreover, relative response factors (RRFs) for the impurities of RIT were also determined.

Results Efficient separation and selectivity were achieved in a shorter run time on an Acquity UPLC BEH Shield RP-18 column (100 mm, 2.1 mm and 1.7 mm) thermostated at 508C using mobile phase A [KH2PO4 buffer ( pH 3.5) : ACN (80 : 20, v/v)] and mobile phase B [ACN : water (80 : 20, v/v)]. The chromatogram was

Table I Chromatographic Performance Data S. no.

Impurity name

RT (min)a

RRTb (n ¼ 6)a

USP resolution (n ¼ 6)a

USP tailing factor (n ¼ 6)a

USP plate count (n ¼ 6)a

RRFc

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 Imp-9 RIT Imp-10 Imp-11 Imp-12 Imp-13

0.528 1.22 2.212 2.796 3.136 4.778 6.178 8.919 10.073 10.323 10.824 11.574 14.704 17.034

0.05 0.12 0.21 0.27 0.30 0.46 0.60 0.86 0.98 1.0 1.05 1.12 1.42 1.65

NA 10.7 12.2 6.7 3.5 14.5 9.5 16 6.6 2.0 3.0 5.0 21.2 14.5

1.1 1.1 1.1 1.1 1.1 1.1 1.0 1.0 1.0 1.1 1.1 1.1 1.0 0.9

2,504 3,834 11,684 15,813 15,144 24,615 21,762 41,446 62,164 58,922 71,709 112,080 143,742 171,696

3.12 0.95 1.03 0.94 2.53 1.50 1.10 0.86 0.58 1.19 0.74 0.96 0.90

a

Mean þ SD (n ¼ 6). RRTs were calculated against the retention time of RIT. RRF was calculated against the response factor of RIT.

b c

monitored at 240 nm with the mobile phase flow rate of 0.5 mL/min using a gradient program (time in min/%B): 0.01/ 17, 0.6/17, 6/30, 11/45, 18/75, 18.01/17 and 20/17. The system suitability parameters were evaluated for RIT and its impurities. The USP tailing factor for all impurities and RIT was found to be ,1.2. The USP resolutions (Rs) between all components are .1.5 (Table I). To obtain the previously optimized chromatographic conditions, the method was developed using different columns, mobile phase pH value, column temperature, flow rate and gradient program as discussed in the ‘Discussion’ section. The method was found to be linear, precise, accurate, robust and stability indicating (Tables II – IV). Three batches of RIT stability samples and NORVIR tablets were analyzed for related compounds and subjected to assay analysis to determine the impurity profile and potency of the drug. The results of the test and stability samples indicated that all of the impurities were present at less than the specification limit in the drug substances and products. The assay results of the RIT stability samples and NORVIR tablets ranged from 98.7 to 100.8% (w/w).

Discussion RIT is synthesized by the key reaction of (2S)-3-methyl2-[[methyl[[2-(1-methylethyl)thiazol-4-yl]methyl]carbamoyl] amino]butanoic acid (Imp-3) with organic coupling reagent to form Imp-3 intermediate (Part A). Thiazol-5-ylmethyl[(1S,2S, 4S)-1-benzyl-4-[[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxy5-phenylpentyl]carbamate (Imp-8) deprotected with HCl to form thiazol-5-ylmethyl (2S,3S,5S)-5-amino-3-hydroxy-1,6-diphe nylhexan-2-ylcarbamate (Imp-2) (Part B). Finally, condensation of Part A with Part B in the presence of a solvent afforded RIT (Figure 2). The potential impurities and degradation products are most likely to arise during the RIT synthesis. As per the article (24), the reported impurities ( phenol, 4-nitrophenol, N-pheno xycarbonyl-L-valine) were observed at the key starting material stage of RIT synthesis. These impurities were well controlled and shown as not detected in the early stage of RIT intermediate synthesis. To monitor the quality of RIT, a single stability-indicating Quantitative Determination of RIT and Its Related Compounds 665

Table II Summary of Forced Degradation Results Degradation condition

Time

Assay (%, w/w) on anhydrous basis

Mass balance (% assay þ % degradation products)

RS by UPLC % degradation

Remarks/observation

Acid hydrolysis (1 N HCl, 708C) Base hydrolysis (0.001 N NaOH, room temperature) Water hydrolysis (708C) Oxidation by 5.0% H2O2 Photodegradation Thermal degradation (908C) solid

5h 30 min 5h 48 h 7 days 10 days

94.2 91.7 93.7 98.9 98.3 99.4

99.5 99.6 99.4 99.2 99.6 99.5

5.30 7.92 5.65 0.47 1.32 0.07

Imp-2, Imp-3 and two unknown degradation products were formed Imp-1 and Imp-9 degradation products were formed Imp-1, Imp-9 and one unknown degradation products were formed No significant degradation observed Two unknown degradation products were formed No significant degradation observed

analytical method able to separate all 13 impurities and the degradation products is required.

Method development The main objective of method development was to achieve simple, rapid and efficient separation between RIT and its related compounds. The main difficulty was to obtain sufficient selectivity and resolution in shorter run time with structurally similar impurities and degradants. To reach final chromatographic conditions, the following chromatographic parameters have been optimized. Wavelength selection RIT and related compounds have similar UV absorption spectra and the absorption maximum observed at 240 nm. A detection wavelength of 240 nm was selected based on the full-range UV spectral data due to its high sensitivity for all related substances and minimal difference in response factors. Evaluation of column stationary phase The structural review depicts the nonpolar nature of RIT and most of its impurities (Figure 1). Hence, we started the initial development activity on different nonpolar stationary phases (C18, C8 and phenyl column) with different selectivities and hydrophobicities (25). The typical retention behaviors of RIT and its impurities at various stationary phases are depicted in Figure 3A. The observations of various column behavior are summarized below. All five columns exhibited a peak tailing factor of 1.0 for RIT. Imp-9 co-eluted with RIT, and Imp-12 and Imp-13 were strongly retained with a HSS T3 column. A BEH C18 column gave co-elution of Imp-9 and RIT with close elution for Imp-10 and Imp-11 (Rs 1.8). In the BEH C8 column, two components such as Imp-4 and Imp-5 were merged together, Imp-9 co-eluted with RIT and close elution for Imp-10 and Imp-11 (Rs 1.0) was noticed. The phenyl column yielded good separation for all components except Imp-9, which closely eluted with RIT (Rs 1.2). Finally, the Polar embedded Waters Acquity BEH Shield RP18 column (100  2.1 mm, 1.7 mm) was evaluated for separation of all impurities and found to be efficient with good resolution between critical pairs, Imp-9 and RIT (Rs 1.8) (Figure 3A). This column was taken for further optimization. Buffer concentration and pH The effect of concentration of KH2PO4 on the separation and retention was studied by varying its concentration from 0.01 to 0.03 M. The buffer concentration showed no effect on the 666 Koppala et al.

retention and resolution RIT and its impurities. The effect of the buffer pH on the retention times of RIT and its impurities was studied from pH 2.0 to 7.0, keeping the other chromatographic parameters unchanged. At pH 5.0, 6.0 and 7.0, the early elution of Imp-3 was observed. This is due to the presence of a hydrophilic ionizable functional group (– COOH). The retention of other impurities and RIT was found to be not effected with buffer pH in the studied range. However, the best retention of early eluting peaks with good resolution was achieved at pH 3.5. Therefore, the mobile phase pH was set at 3.5 for the separation of all impurities, which also ensures good column performance as operated in acidic pH. Evaluation of organic solvent type Initially, methanol was used as an organic modifier. The retention time for all impurities was increased along with poor resolution among the peaks that were observed. To improve the resolution with considerable retention time, ACN was tried as an organic modifier. The resolution among all impurities was improved with considerable reduction in retention time. Improved response with better peak shape and base line was observed with ACN. Hence, ACN was selected as organic modifier. Evaluation of mobile phase and gradient program The RIT impurities blend solution was subjected to separation by reversed-phase LC on a Waters Acquity BEH Shield RP-18, 100  2.1 mm, 1.7 mm column with buffer and ACN in the ratio of 60 : 50 and 50 : 50 (v/v) with isocratic flow. Six compounds, that is Imp-1 and Imp-2, Imp-3 and Imp-4 and Imp-5 and Imp-6, were closely eluted, Imp-9 was co-eluted with RIT and also Imp-12 and Imp-13 were strongly retained. RIT impurities have both hydrophilic (Imp-1, Imp-2 and Imp-3) and hydrophobic (Imp-12 and Imp-13) attributes. Because the impurities in the mixture have a wide range of polarities, the need for a gradient run was assessed using mobile phase A: 0.01 M KH2PO4 buffer ( pH 3.5) and mobile phase B [ACN : water (80 : 20, v/v)]. The initial gradient run [(time in min)/%B; 0/0 and 30/100] provided an estimate of the percentage of organic ratio and approximate retention times for the impurities. The retention times of the first and last impurity peaks were 0.5 and 26 min, respectively. To minimize the run time with better separations, many attempts were made with different organic solvent compositions in mobile phases A and B, along with different gradient elutions (Figure 3B). It was found that use of phosphate buffer ( pH 3.5), ACN in the ratio of 80 : 20 (v/v) as mobile phase A; water, ACN in the ratio of 20 : 80 (v/v) as mobile phase B, with gradient elution (time (min)/%B) 0.01/17, 0.6/17, 6/30, 11/45, 18/75,

0.9998 3,890,750 290.03 21.6 0.9996 0.9998 6,635,851 2133.68 21.3 0.9996 0.9999 10,699,077 2150.81 20.6 0.9998 1.0000 4,076,305 9.36 0.1 0.9999 0.9998 4,515,418 250.17 20.7 0.9997 0.9996 4,183,994 299.16 21.4 0.9994 0.9999 12,937,980 300.71 1.0 0.9999

Linearity range is LOQ 150% with respect to 0.15% specification level for Imp-1, Imp-2, Imp-5, Imp-7 and Imp-10; 0.1% specification level for Imp-3, Imp-4, Imp-6, Imp-8, Imp-9, Imp-11 and Imp-13 and 0.2% specification level for Imp-12; RSD, relative standard deviation. a LOQ precision. b Repeatability. c Intermediate precision.

0.9995 4,240,123 253.29 21.1 0.9991 0.9998 4,046,422 245.89 20.4 0.9995 0.9994 3,240,858 6.38 0.1 0.9991 1.0000 5,130,655 229.47 20.2 0.9999 0.9997 4,423,279 36.84 0.5 0.9996

6.70

1.0000 4,564,578 239.87 20.4 0.9999

0.9995 2,641,824 31.04 0.8 0.9994

0.10 0.122 0.416 104.1 5.30 0.38 0.38 99.2 98.0 97.6 0.10 0.138 0.470 96.0 6.71 0.28 0.71 102.9 96.7 101.4 0.10 0.137 0.471 100.2 4.84 1.53 0.44 101.7 98.9 101.3 0.10 0.084 0.286 103.1 2.32 0.73 0.45 105.8 103.7 103.1 0.15 0.037 0.125 98.8 1.77 0.29 0.40 103.8 103.4 102.1 0.15 0.078 0.265 103.0 2.48 0.39 0.31 101.5 101.0 100.0 0.15 0.014 0.050 103.2 4.16 0.14 0.16 95.9 95.8 97.5

Specifications (%) LOD (mg/mL) LOQ (mg/mL) LOQ accuracy % RSD (n ¼ 6)a % RSD (n ¼ 6)b % RSD (n ¼ 6)c Accuracy at 50% Accuracy at 100% Accuracy at 150% Regression equation (y) Correlation Slope (b) Intercept (a) % Y-intercept R 2 value

0.10 0.102 0.349 97.1 3.03 0.72 0.53 100.4 105.1 104.2

0.10 0.125 0.419 97.3 2.45 0.53 0.76 101.2 104.7 103.3

0.15 0.108 0.370 104.0 3.27 0.52 0.58 100.5 98.3 98.6

0.10 0.165 0.570 97.9 6.11 2.01 0.47 103.8 105.0 102.0

0.112 0.381

0.15 0.113 0.381 99.6 5.43 0.54 0.77 96.4 99.0 99.3

0.20 0.131 0.444 98.1 4.60 0.91 0.91 97.3 99.6 100.2

Imp-13 Imp-12 Imp-11 Imp-10 RIT Imp-9 Imp-8 Imp-7 Imp-6 Imp-5 Imp-4 Imp-3 Imp-2 Imp-1 Validation parameter

Table III Summary of Method Validation for RIT and Its Impurities

18.01/17 and 20/17 enabled separation for all components with good peak shape, resolution and retention (Figure 3B). Evaluation of column temperature and flow rate In all attempts, Imp-9 closely eluted with RIT (Rs 1.0) to increase resolution column temperature, and the mobile phase flow rate was also studied. Upon increasing the column oven temperature from 25 to 358C, a little separation was observed. The column oven temperature optimized to 508C. At this temperature, the resolution between Imp-9 and RIT was increased to 1.5. By adjusting the mobile phase flow rate from 0.3 to 0.5 mL/min while keeping the column oven temperature at 508C, base-to-base separation was observed between Imp-9 and RIT. The resolution was increased to 2.0. Hence, the chromatographic column oven temperature and mobile phase flow rate were set as 508C and 0.5 mL/min, respectively. The resulting chromatogram is depicted in Figure 3C. Evaluation of diluent and filter RIT is very sensitive to alkali. At basic pH, RIT drastically degraded into Imp-1 and Imp-9. Hence, the diluent was set as acidic buffer ( pH 3.5; 0.01 M KH2PO4) and ACN in the ratio of 1 : 1. This diluent was found to be more suitable for RIT and its impurities. Placebo interference was also verified and found that no interference was observed at the retention time of any of the impurities and RIT. The filter (Millipore nylon membrane) interference was checked and found that no peaks observed at the retention time of RIT and its impurities. Relative response factor RRFs were established for all known impurities as the ratio of the slope of impurities and the slope of RIT. The slope value obtained with the linear calibration plot was used for the determination of RRF (Table I).

Forced degradation studies Hydrolysis

Acid hydrolysis. RIT was subjected to 1 N HCl at room temperature for 48 h, but no significant degradation was observed. To get considerable degradation, RIT was refluxed with 1 N HCl at 708C, where 5.3% degradation was observed in 5 h. Under acid hydrolysis, four degradation products such as Imp-2 [m/z 426.2; relative retention time (RRT)  0.12], Imp-3 (m/z 314.2; RRT  0.21), unknown Imp-1 (m/z 721; RRT  0.64) and unknown Imp-2 (m/z 721; RRT  0.88) were formed (Figure 4A). The known impurities Imp-2 and Imp-3 are generated due to the hydrolysis of amide function of RIT to amine and acid, respectively, and the pathway of degradation depicted in Figure 5A. Base hydrolysis. In the initial experiment, RIT was subjected to 1 N NaOH solution at room temperature for 1 h, which showed complete degradation of RIT into Imp-1 (m/z 116.2; RRT  0.05) and Imp-9 (m/z 606.6; RRT  0.98) (Figure 4B). Gradually, the concentration of the base was reduced to 0.001 N NaOH, where 7.9% degradation was observed in 30 min at room temperature. RIT was found to be very sensitive toward base hydrolysis, where the carbamate function of RIT cyclizes with Quantitative Determination of RIT and Its Related Compounds 667

Table IV Results of Robustness Study Parameter/variation

USP resolution (Rs) Imp-1

As such conditions Organic solvent 95% 105% Mobile phase pH pH 3.3 pH 3.7 Flow rate (mL/min) 0.45 0.55 Column temperature (8C) 45 55

Imp-2

Imp-3

Imp-4

Imp-5

Imp-6

Imp-7

Imp-8

Imp-9

RIT

Imp-10

Imp-11

Imp-12

Imp-13

10.7

12.2

6.7

3.5

14.5

9.5

16.0

6.6

2.0

3.0

5.0

21.2

14.5

11.5 9.8

11.2 10.9

8.7 7.7

3.4 4.4

16.7 14.7

9.2 9.5

18.4 17.7

7.2 7.1

1.9 1.5

3.3 3.3

5.7 5.6

21.2 22.3

16.4 15.9

9.3 10.1

12.0 9.5

6.3 9.1

4.6 4.2

14.4 14.9

9.6 9.8

17.8 18.1

7.0 7.1

1.8 1.8

3.3 3.2

5.7 5.7

21.5 21.5

15.4 15.6

9.6 9.1

10.5 9.6

8.1 7.8

5.5 4.0

13.7 14.2

11.0 7.2

17.3 14.8

7.0 7.0

1.7 1.9

3.4 3.1

5.4 6.4

24.5 22.0

16.9 15.5

9.7 9.0

9.9 10.1

8.3 7.6

5.5 3.8

13.3 14.6

7.4 10.6

15 17.6

6.5 7.4

1.6 2.1

3.3 3.2

5.6 5.2

19.5 22.0

15.9 15.5

Figure 2. Reaction scheme for the synthesis of RIT.

the adjacent hydroxyl group resulting in alcoholic moiety (Imp-1) and cyclic product (Imp-9) (Figure 5B).

Water hydrolysis. RIT was refluxed with water at 708C for 5.0 h, and 5.6% degradation was observed, where Imp-1 (m/z 116.2; RRT  0.05), Imp-9 (m/z 606.6; RRT  0.98) and unknown impurity (m/z 551; RRT  0.62) were found as degradants (Figure 4C). Under aqueous hydrolysis, the carbamate function cyclizes with the adjacent hydroxyl group producing alcoholic moiety (Imp-1) and cyclic product (Imp-9). The unknown impurity (m/z 551) was formed due to nucleophilic attack of water on the amide functional group followed by cyclization as depicted in Figure 5C. The unknown impurity (m/z 551) structure was proposed based on the LC–MS-MS fragmentation pattern as depicted in Figure 6A. 668 Koppala et al.

Oxidative degradation RIT was subjected to oxidative stress using 5.0% H2O2 at room temperature for 48 h. No considerable degradation (0.4%) was noticed (Figure 4D). Thermolytic and photolytic degradation The drug RIT in the solid form was exposed to the thermolytic (908C for 7 days) and photolytic (an overall illumination of 1.2 million lux-hours for light and 200 W h/m2 for UV region) stress. No degradation was observed at the thermolytic condition. In photolytic degradation, two unknown degradation products such as unknown imp-1 (m/z 737; RRT  0.58) and unknown imp-2 (m/z 753; RRT  0.68) were formed (Figure 4E). The structures (m/z 737 and 753) were proposed based on the LC – MS-MS fragmentation pattern as depicted in Figure 6B and C.

Figure 3. Method development chromatograms. (A) Selectivity differences of RIT and its impurities by using different stationary phases. (B) Retention of RIT and its impurities with gradient elusion. (C) Blend chromatogram of RIT and its related impurities in final chromatographic conditions.

The isopropyl moiety in the thiazole ring of RIT is susceptible to radical mechanism in the presence of UV light and atmospheric oxygen (26). In the presence of UV light, the oxygen molecule abstracts a hydrogen atom from the tertiary carbon of the isopropyl moiety, which produces a RIT tertiary alkyl radical and a hydroperoxide radical. These two radicals interact

with each other to form the RIT hydroperoxide as impurity (m/z 753). The RIT hydroperoxide molecule further decomposes into a RIT alkoxy radical and a hydroxyl radical. The RIT alkoxy radical abstracts hydrogen from another molecule of RIT and produces RIT hydroxide as degradant (m/z 737) (Figure 5D). Quantitative Determination of RIT and Its Related Compounds 669

Figure 4. Typical chromatograms of RIT under stress conditions: (A) acid hydrolysis, (B) base hydrolysis, (C) water hydrolysis and (D) photodegradation.

670 Koppala et al.

Figure 5. Degradation mechanism for RIT under forced degradation studies. (A) Acid hydrolysis degradation mechanism and formation of Imp-2 and Imp-3. (B) Base hydrolysis degradation mechanism and formation of Imp-1 and Imp-9. (C) Water hydrolysis degradation mechanism and formation of Imp-1, Imp-9 and an unknown degradant (m/z 551). (D) Photodegradation mechanism by atmospheric oxygen and formation of an unknown degradant (m/z 737, 753).

Stressed samples of acid, base, water hydrolysis, peroxide and photolytic reactions were analyzed by LC –ESI-MS in the positive ionization mode. The UPLC chromatograms of stressed samples were compared with the HPLC – MS chromatograms, and the degradation peaks were correlated with the LC – MS peaks

based on the order of elution, % level of degradation and UV spectral data. The results from the peak purity assessment revealed that the purity angle was less than the purity threshold in all of the stressed samples, indicating peak homogeneity. The principal Quantitative Determination of RIT and Its Related Compounds 671

Figure 5. Continued

Figure 6. Identification of unknown degradation products (m/z 551, 737 and 753) by using LC –MS-MS analysis. (A) LC– MS-MS fragment compounds of unknown degradant (m/z 551) of water hydrolysis. (B) LC– MS-MS fragment compounds of unknown photo degradant (m/z 753) of photolytic degradation. (C) LC –MS-MS fragment compounds of unknown photo degradant (m/z 737) of photolytic degradation.

peak m/z value [MþH]þ 721.6 (molecular weight 720.95 g/mol) under all stress conditions supported the identification of RIT. The mass balance (% assay þ % impurities þ % degradation

672 Koppala et al.

products) was calculated for all of the stressed samples and found to be close to 99.0% (Table II), indicating the stability-indicating nature of the developed UPLC method.

Figure 6. Continued

Method validation Validation of the chromatographic method was performed with respect to sensitivity, precision, linearity, accuracy, solution stability and robustness. Sensitivity The LOD and LOQ values for all impurities were determined by injecting a series of diluted solutions with known concentration to obtain the signal-to-noise (S/N) ratio values of 3 and 10, respectively. For LOQ, the S/N ratios for all the impurities were ranging from 9.7 to 10.3. A precision study was performed at the LOQ level by injecting six individual preparations of RIT and impurities and calculated the % relative standard deviation (RSD) for the areas of each peak. The RSDs were found to be between 1.77 and 6.71%. Accuracy at the LOQ level was verified by injecting three individual preparations of RIT spiked with impurities at the LOQ level. The percent recoveries were calculated for each impurity and those are ranging from 96.0 to 104.1%. The results were in the range of 0.014–0.165 mg/mL for LOD and 0.05 –0.570 mg/mL for LOQ (Table III). Precision The repeatability and ruggedness of the method were performed by six individual determinations of RIT (1,500 mg/mL) by spiking

with impurities at the specification level. The ruggedness was determined by repeating the same experiment on two different days by different analysts using different equipment. The % RSD was calculated for each impurity (Table III). The precision of the assay was evaluated by performing six (n ¼ 6) independent assays of the RIT test sample against the qualified reference standard. The assay results obtained on the two different days (n ¼ 6) were 99.85 + 0.18 and 99.75 + 0.32 (mean + RSD). These results confirmed the high precision. Linearity Linearity test solutions were prepared from impurity stock solution at seven different concentration levels ranging from LOQ to 150% of the specification level (i.e., LOQ, 0.900, 1.125, 1.688, 2.250, 2.813 and 3.375 mg/mL for Imp-1, Imp-2, Imp-5, Imp-7 and Imp-10; LOQ, 0.600, 0.750, 1.125, 1.50, 1.875 and 2.250 mg/mL for RIT, Imp-3, Imp-4, Imp-6, Imp-8, Imp-9, Imp-11 and Imp-13 and LOQ, 1.200, 1.500, 2.250, 3.00, 3.750 and 4.500 mg/mL for Imp-12). The calibration curve was drawn by plotting the impurity area versus the concentration. The correlation coefficient obtained was .0.999 for all the impurities (Table III). Similarly, the linearity of RIT in the assay method was also established at six concentrations (from 75 to 450 mg/mL). The correlation coefficient (r) was .0.999. Quantitative Determination of RIT and Its Related Compounds 673

Figure 6. Continued

Accuracy Accuracy of the method was evaluated by spiking with known amounts of impurities to the placebo-based solution of the test sample (1,500 mg/mL) at the level of 50, 100 and 150% of specification in triplicate. Similarly, the accuracy of the RIT determination in the assay method was evaluated in triplicate at three concentration levels (150, 300 and 450 mg/mL). The percent recoveries were calculated for related substances and those are ranging from 96.0 to 105.0% (Table III). The percent recovery of RIT in bulk drug sample was ranging from 98.0 to 101.0% (w/w) in its assay method. Solution stability and mobile phase stability The solution stability and mobile phase stabilities at 258C temperature were evaluated by injecting the test solutions spiked with impurities daily for up to 5 days. Prepared mobile phase was kept constant during the study period. No significant changes in the amounts of impurities were observed during the study. The % RSD values for the RIT assay during the solution and mobile phase stability experiments were within 0.6%. These results confirmed that sample solution and mobile phase were stable up to 5 days at ambient temperature. 674 Koppala et al.

Robustness To determine the robustness of the method, experimental conditions were deliberately altered. The factors chosen for this study, which were the critical sources of variability in the operating procedures such as flow rate (0.5 + 0.05 mL/min), mobile phase pH (3.5 + 0.2), mobile phase composition (mobile phase A +5% ACN; mobile phase B +5% ACN) and column oven temperature (50 + 58C) were identified. Resolution between RIT and impurities was evaluated in the deliberately altered experimental conditions. The method was robust in the specified range (Table IV).

Conclusion A simple and selective stability-indicating gradient RP-UPLC method has been developed for the quantitative determination of RIT and its impurities in drug substance and drug product. This method is capable of separating all impurities with good resolution within 20 min. This method exhibited excellent performance in terms of sensitivity and speed. Forced degradation studies were conducted, and the major degradants were identified using LC–MS-MS. A plausible mechanism for the formation

of degradation impurities was proposed based on the known reactivity of the drug through hydrolysis and photolysis. The developed method was validated as per the ICH guidelines and found to be specific, precise, accurate and linear. Thus, the method can be used for quality control analysis of drug substance and pharmaceutical dosage forms.

Acknowledgments The authors thank the management of Dr Reddy’s group for supporting this work. They also acknowledge the Process Research Group for providing the samples for this research.

References 1. Nageswara Rao, R., Ramachandra, B., Mastan Vali, R., Satyanarayana Raju, S.; LC – MS/MS studies of ritonavir and its forced degradation products; Journal of Pharmaceutical and Biomedical Analysis, (2010); 53: 833–842. 2. Yekkala, R.S., Ashenafi, D., Marie¨n, I., Xin, H., Haghedooren, E., Hoogmartens, J., et al.; Evaluation of an International Pharmacopoeia method for the analysis of ritonavir by liquid chromatography; Journal of Pharmaceutical and Biomedical Analysis, (2008); 48: 1050–1054. 3. Granda, B.W., Giancarlo, G.M., Moltke, L.L., Greenblatt, D.J.; Analysis of ritonavir in plasma/serum and tissues by high-performance liquid chromatography; Journal of Pharmacological and Toxicological Methods, (1998); 40: 235– 239. 4. Akeb, F., Ferrua, B., Creminon, C., Roptin, C., Grassi, J., Nevers, M.C., et al.; Quantification of plasma and intracellular levels of the HIV protease inhibitor ritonavir by competitive ELISA; Journal of Immunological Methods, (2002); 263: 1 –9. 5. Janoly, A., Bleyzac, N., Favetta, P., Gagneu, M.C., Bourhis, Y., Coudray, S., et al.; Simple and rapid high-performance liquid chromatographic method for nelfinavir, M8 nelfinavir metabolite, ritonavir and saquinavir assay in plasma; Journal of Chromatography B, (2002); 780: 155–160. 6. Sarasa-Nacentaa, M., Lo´pez-Pu´aa, Y., Mallolas, J., Blanco, J.L., Gatell, J.M., Carne, X.; Simultaneous determination of the HIV-protease inhibitors indinavir, amprenavir, ritonavir, saquinavir and nelfinavir in human plasma by reversed-phase high-performance liquid chromatography; Journal of Chromatography B, (2001); 757: 325– 332. 7. Marzolini, C., Telenti, A., Buclin, T., Biollaz, J., Decosterd, L.A.; Simultaneous determination of the HIV protease inhibitors indinavir, amprenavir, saquinavir, ritonavir, nelfinavir and the non-nucleoside reverse transcriptase inhibitor efavirenz by high-performance liquid chromatography after solid-phase extraction; Journal of Chromatography B, (2000); 740: 43 –58. 8. Albert, V., Modamio, P., Lastra, F.C., Eduardo, M.L.; Determination of saquinavir and ritonavir in human plasma by reversed-phase highperformance liquid chromatography, and the analytical error function; Journal of Pharmaceutical and Biomedical Analysis, (2004); 36: 835– 840. 9. Jagadeeswaran, M., Gopal, N., Pavan Kumar, K., Sivakumar, T.; Quantitative estimation of lopinavir and ritonavir in tablet dosage forms by RP-HPLC method; American Journal of PharmTech Research, (2012); 2: 576– 583.

10. Sumeetha, A., Kathirvel, S., Ramachandrika, G.; A validated RP-HPLC method for simultaneous estimation of lopinavir and ritonavir in combined dosage form; International Journal of Pharmacy and Pharmaceutical Sciences, (2011); 3: 49 –51. 11. Varaprasad, B.L., Harinadha, B.K., Ravikumar, A., Vijaykumar, G.; Development method validation of RP-HPLC method for simultaneous determination of lopinavir and ritonavir in bulk and formulation dosage; International Research Journal of Pharmaceutical and Applied Sciences, (2012); 2: 84– 90. 12. Usami, Y., Oki, T., Nakai, M., Sagisaka, M., Kaneda, T.; A simple HPLC method for simultaneous determination of lopinavir, ritonavir and efavirenz; Chemical and Pharmaceutical Bulletin, (2003); 51: 715– 718. 13. Phechkrajang, C.M., Thin, E.E., Sratthaphut, L., Nacapricha, D., Wilairat, P.; Quantitative determination of lopinavir and ritonavir in syrup preparation by liquid chromatography; Mahidol University Journal of Pharmaceutical Science, (2009); 36: 1 –12. 14. Chiranjeevi, K., Channabasavaraj, K.P.; Development and validation of RP-HPLC method for quantitative estimation of ritonavir in bulk and pharmaceutical dosage forms; International Journal of Pharmaceutical Sciences and Research, (2011); 2: 596– 600. 15. Donato, E.M., Dias, C.L., Rossi, R.C., Valente, R.S., Fro¨ehlich, P.E., Bergold, A.M.; LC method for studies on the stability of lopinavir and ritonavir in soft gelatin capsules; Chromatographia, (2006); 63: 437– 443. 16. Behera, A., Moitra, S.K., Chandra, S.S., Meher, A.K., Parida, A., Gowri Sankar, D.; Method development, validation and stability study of ritonavir in bulk and pharmaceutical dosage form by spectrophotometric method; Chronicles of Young Scientists, (2011); 2: 161 – 167. 17. Seetaramaih, K., Anton Smith, A., Ramyateja, K., Alagumanivasagam, G., Manavalan, R.; Spectrophotometric determination of ritonavir in bulk and pharmaceutical formulation; Scientific Reviews & Chemical Communications, (2012); 2: 1 –6. 18. Sudha, T., Vanitha, R., Ganesan, V.; Development and validation of RP-HPLC and HPTLC methods for estimation of ritonavir in bulk and in pharmaceutical formulation; Der Pharma Chemica, (2011); 3: 127–134. 19. Patel, D.J., Desai, S.D., Savaliya, R.P., Gohil, D.Y.; Simultaneous HPTLC determination of lopinavir and ritonavir in combined dosage form; Asian Journal of Pharmaceutical and Clinical Research, (2011); 4: 59– 61. 20. Ritonavir Monograph, United States Pharmacopeia; 30th ed. United States Pharmacopeial Convention, Rockville, MD, USA, USP-35, NF-30, S2, pp. 5991. 21. Ritonavir Monograph, Pharmeuropa, 17th vol. European Directorate for the Quality of Medicines, Strasbourg, France, (2005), pp. 498–502. 22. ICH Q2 (R1); Validation of analytical procedures: text and methodology; (2005). 23. ICH Q1A (R2); Stability testing of new drug substances and products; (2003). 24. Venugopal, N., Vijaya Bhaskar Reddy, G., Madhavi, G.; Development and validation of a systematic UPLC – MS/MS method for simultaneous determination of three phenol impurities in Ritonavir; Journal of Pharmaceutical and Biomedical Analysis, (2014); 90: 127–133. 25. Waters Reversed-Phase Selectivity Chart. http://www.waters.com/ waters/promotionDetail.htm?id=10048475 (accessed June, 2013). 26. Ranby, B., Lucki, J.; New aspects of photodegradation and photooxidation of polystyrene; Pure and Applied Chemistry, (1980); 52: 295–303.

Quantitative Determination of RIT and Its Related Compounds 675