L. K. Ravindranath

FORCED DEGRADATION STUDIES: PRACTICAL APPROACH - OVERVIEW OF REGULATORY GUIDANCE AND LITERATURE FOR THE DRUG PRODUCTS AND DRUG SUBSTANCES

The Objective of the review article is to give a detailed description of the forced degradation studies as per the regulatory guidelines that are associated with various regulatory agencies. This article summarizes the collective views of industry practices on the topic of forced degradation studies. The article includes an overview of existing guidance’s and literature for best practices.

Simultaneous estimation of amlodipine and atenolol in human plasma: a sensitive LC–MS/MS method validation and its application to a clinical PK study

BACKGROUND A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for simultaneous quantification of amlodipine (AMD) and atenolol (ATL) in human plasma (200 µl) using AMD-d4 and ATL-d7, respectively, as an internal standard (IS) as per the regulatory guidelines. RESULTS The SPE method was used to extract the analytes and IS from human plasma. The chromatographic resolution of AMD, ATL and corresponding IS was achieved using an isocratic flow on a C18 column. The total chromatographic run time was 3 min. A linear response function was established for the range of concentrations 50-8000 pg/ml and 10-800 ng/ml for AMD and ATL, respectively, in human plasma. CONCLUSION The intra- and inter-day accuracy and precision values for AMD and ATL met the acceptance as per regulatory guidelines. The validated assay was applied to a fixed-dose combination of AMD and ATL (Adopin-AT(®)) PK study in humans.

Determination of the quaternary ammonium compound trospium in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A highly sensitive, specific and evaporation free SPE extraction, LC-MS/MS method has been developed for the estimation of trospium in human plasma using trospium-d8 as an internal standard (IS). The analyte was separated using isocratic mobile phase on reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M(+)] cations, m/z 392-164 for trospium and m/z 400-172 for the IS. The total run time was 3.50 min and the elution of trospium and trospium-d8 (IS) occurred at 2.8 min. The developed method was validated in human plasma with a lower limit of quantification of 0.05 ng/mL. A linear response function was established for the range of concentrations 0.05-10 ng/mL (r>0.998) for trospium in human plasma. The intra- and inter-day precision values for trospium met the acceptance as per FDA guidelines. Trospium was stable in the battery of stability studies viz., bench-top, auto-sampler, dry extracts and freeze/thaw cycles. The developed assay method was applied to an oral pharmacokinetic study in humans.

Development and validation of a highly sensitive LC-MS/MS method for quantitation of dexlansoprazole in human plasma: application to a human pharmacokinetic study

A highly sensitive, specific and simple LC-MS/MS method was developed for the simultaneous estimation of dexlansoprazole (DEX) with 50 μL of human plasma using omeprazole as an internal standard (IS). The API-4000 LC-MS/MS was operated under multiple reaction-monitoring mode using electrospray ionization. A simple liquid-liquid extraction process was used to extract DEX and IS from human plasma. The total run time was 2.00 min and the elution of DEX and IS occurred at 1.20 min. This was achieved with a mobile phase consisting of 0.2% ammonia-acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on an X-terra RP 18 (50 × 4.6 mm, 5 µm) column. The developed method was validated in human plasma with a lower limit of quantitation of 2 ng/mL for DEX. A linear response function was established for the range of concentrations 2.00-2500.0 ng/mL (r > 0.998) for DEX. The intra- and inter-day precision values for DEX met the acceptance criteria as per FDA guidelines. DEX was stable in the battery of stability studies, viz. bench-top, auto-sampler and freeze-thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.

A rapid LC-MS/MS method for quantitation of eszopiclone in human plasma: application to a human pharmacokinetic study.

A highly reproducible, specific and cost-effective LC-MS/MS method was developed for simultaneous estimation of eszopiclone (ESZ) with 50 μL of human plasma using paroxetine as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract ESZ and IS from human plasma. The total run time was 1.5 min and the elution of ESZ and IS occurred at 0.90 min; this was achieved with a mobile phase consisting of 0.1% formic acid-methanol (15:85, v/v) at a flow rate of 0.50 mL/min on a Discover C(18) (50 × 4.6 mm, 5 µm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.1 ng/mL for ESZ. A linear response function was established for the range of concentrations 0.10-120 ng/mL (r > 0.998) for ESZ. The intra- and inter-day precision values for ESZ were acceptable as per FDA guidelines. Eszopiclone was stable in the battery of stability studies, viz. bench-top, autosampler and freeze-thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.

Development and validation of a highly sensitive LC‐MS/MS method for simultaneous quantitation of nortriptyline and 10‐hydroxynortriptyline in human plasma: application to a human pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed for simultaneous estimation of nortriptyline (NTP) and 10-hydroxynortriptyline (OH-NTP) in human plasma (250 µL) using carbamazepine as an internal standard (IS). LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract NTP, OH-NTP and IS from human plasma. The total run time was 2.5 min and the elution of NTP, OH-NTP and IS occurred at 1.44, 1.28 and 1.39 min, respectively; this was achieved with a mobile phase consisting of 20 mm ammonium acetate : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPURITY C(18) column. The developed method was validated in human plasma with a lower limit of quantitation of 1.09 ng/mL for both NTP and OH-NTP. A linear response function was established for the range of concentrations 1.09-30.0 ng/mL (r > 0.998) for both NTP and OH-NTP. The intra- and inter-day precision values for NTP and OH-NTP met the acceptance as per FDA guidelines. NTP and OH-NTP were stable in a battery of stability studies, i.e. bench-top, auto-sampler and freeze-thaw cycles. The developed assay was applied to a pharmacokinetic study in humans.

Rapid quantification of amlodipine enantiomers in human plasma by LC-MS/MS: application to a clinical pharmacokinetic study

A rapid, simple, specific and sensitive LC-MS/MS method has been developed and validated for the enantiomeric quantification of amlodipine (AML) isomers [R-amlodipine (R-AML) and S-amlodipine (S-AML)] with 200 μL of human plasma using R-AML-d4 and S-AML-d4 as corresponding internal standards as per regulatory guidelines. A simple liquid-liquid extraction process was used to extract these analytes from human plasma. The total run time was 3.5 min and the elution of R-AML, S-AML, R-AML-d4 and S-AML-d4 occurred at 1.62, 2.51, 1.63 and 2.53 min, respectively. This was achieved with a mobile phase consisting of 0.2% ammonia-acetonitrile (20:80, v/v) at a flow rate of 1 mL/min on a Chiralcel OJ RH column. A linear response function was established for the range of concentrations 0.1-10 ng/mL (r >0.998) for each enantiomer. The intra- and inter-day precision values for both enantiomers met the acceptance criteria. Both enantiomers were stable in a set of stability studies, viz. bench-top, auto-sampler, freeze-thaw cycles and long-term. The current assay was successfully applied to a pharmacokinetic study to quantitate AML enantiomers following oral administration of 10 mg AML tablet to humans.

Validation and clinical application of an LC‐ESI‐MS/MS method for simultaneous determination of tolmetin and MED5, the metabolites of amtolmetin guacil in human plasma

A highly sensitive, rapid assay method has been developed and validated for the simultaneous estimation of tolmetin (TMT) and MED5 in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. A simple solid-phase extraction process was used to extract TMT and MED5 along with mycophenolic acid (internal standard, IS) from human plasma. Chromatographic separation was achieved with 0.2% formic acid-acetonitrile (25:75, v/v) at a flow rate of 0.50 mL/min on an X-Terra RP(18) column with a total run time of 2.5 min. The MS/MS ion transitions monitored were 258.1 → 119.0 for TMT, 315.1 → 119.0 for MED5 and 321.2 → 207.0 for IS. Method validation and clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 20 ng/mL and the linearity was observed from 20 to 2000 ng/mL, for both the anlaytes. The intra-day and inter-day precisions were in the range 3.27-4.50 and 5.32-8.18%, respectively for TMT and 4.27-5.68 and 5.32-8.85%, respectively for MED5. This novel method has been applied to a clinical pharmacokinetic study.

LC and LC-MS/MS study of forced decomposition behavior of anastrozole and establishment of validated stability-indicating analytical method for impurities estimation in low dose anastrozole tablets.

Anastrozole tablets were subjected to different ICH prescribed stress conditions of thermal, hydrolysis, humidity, photolysis and oxidation stress. The drug was found to be stable for all the stressed conditions except for oxidation. Separation of anastrozole from its potential impurities, degradation products and five anastrozole related compounds as main impurities were achieved on Inertsil ODS-3V, 250 mm x 4.6 mm i.d, 5 microm analytical column using reversed phase high performance liquid chromatography (RP-HPLC). The elution of impurities employed time dependent gradient programmed mobile phase consisting of water as mobile phase-A and acetonitrile as mobile phase-B at column flow rates of 1 ml/min and at 215 nm UV detection. The same method was also extended to LC-MS/MS studies which were carried out to identify the degradation product. The method developed was established to have sufficient intermediate precision as similar separation was achieved on another instrument handled by a different operator. The LOQ for anastrozole related compound-A (RC-A), related compound-B (RC-B), related compound-C (RC-C), related compound-D (RC-D), related compound-E (RC-E) and anastrozole were 0.05, 0.03, 0.03, 0.06, 0.06 and 0.06 microg ml(-1) respectively. The linearity of the proposed method for all the above related compounds was investigated in the range of LOQ to 0.600 microg ml(-1) respectively. The specificity was established through peak purity testing using a photo-diode array detector. Method was validated according to ICH guidelines and statistical analysis of the data proved to be suitable for stability testing at quality control.

Regulatory Requirement – Validated, Specific, and Stability Indicating Analytical Method for Zoledronic Acid and Its Related Impurities by Ion Pair Reversed Phase Liquid Chromatography (IP-RPLC)

Abstract A. sensitive and precise ion pair reversed phase liquid chromatographic (IP-HPLC) method was developed and validated for the determination of zoledronic acid in the presence of its impurities and degradants in parental sterile dosage form. The chromatograph employs octadecyl silane chemically bonded to porous silica, stainless steel analytical column with mobile phase of methanol, and buffer at column flow rate of 0.8 mL min−1, temperature thermo stated to 50°C, and a UV-vis detector wavelength of 215 nm. The buffer used in the mobile phase contains phosphate buffer and tetra butyl ammonium hydrogen sulphate as ion pair, and the pH of the buffer adjusted to 7.2 with dilute orthophosphoric acid. The proposed method was established to have sufficient intermediate precision as a similar separation was achieved on another instrument handled by a different operator, and the method was validated for precision, specificity, linearity, range, accuracy, LOQ, ruggedness, and robustness. The method was also successful in the analysis of the drug in marketed samples subjected to stability testing under stressed conditions of pH hydrolysis, oxidation, and temperature stress as per ICH guidelines. Statistical analysis of the data proved that the method is precise and accurate and can be applied for quantification of impurities and actives in pharmaceutical dosage formulation.