Liquid Chromatographic Assay Method Research Articles

A validated stability indicating ion-pair RP-LC method for zoledronic acid

The present paper describes the development of a stability indicating high performance liquid chromatographic (HPLC) assay method for zoledronic acid in the presence of its impurities and degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The degradation of zoledronic acid was observed under oxidative stress at higher temperature. The drug was found to be stable in other stress conditions attempted. Successful separation of the drug from the degradation products formed under stress conditions was achieved on a C18 column using a mixture of phosphate buffer that contains 7 mM tetra butyl ammonium hydrogen sulphate, an ion-pairing agent and methanol (95:5) as mobile phase. The developed HPLC method was validated with respect to response function, precision, accuracy, specificity and robustness. The developed HPLC method to determine the related substances and assay determination of zoledronic acid can be used to evaluate the quality of regular production samples. It can be also used to test the stability samples of zoledronic acid.

A stability indicating LC method for zolmitriptan

A gradient, reversed-phase liquid chromatographic (RP-LC) assay method was developed for the quantitative determination of zolmitriptan, used to treat severe migraine headaches. The developed method is also applicable for the related substances determination in bulk drugs. The chromatographic separation was achieved on a Waters X Terra RP18, 250 mm × 4.6 mm, 5 μm column. The gradient LC method employs solutions A and B as mobile phase. The solution A contains a mixture of phosphate buffer pH 9.85:methanol:acetonitrile (70:20:10, v/v/v) and solution B contains a mixture of phosphate buffer, pH 9.85:acetonitrile (30:70). The flow rate was 1.0 ml/min and the detection wavelength was 225 nm. In the developed HPLC method, the resolution between zolmitriptan and its potential impurities, namely Imp-1, Imp-2 and Imp-3 was found to be greater than 3. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in alkaline medium and oxidative stress conditions. Degradation product formed during base hydrolysis was found to be Imp-3. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.5%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.

ICH guidance in practice: establishment of inherent stability of secnidazole and development of a validated stability-indicating high-performance liquid chromatographic assay method

The degradation behaviour of secnidazole was investigated under different stress degradation (hydrolytic, oxidative, photolytic and thermal) conditions recommended by International Conference on Harmonisation (ICH) using HPLC and LC–MS. A stability-indicating HPLC method was developed that could separate drug from degradation products formed under various conditions. Secnidazole was found to degrade significantly in alkaline conditions, oxidative stress, and also in the presence of light. Mild degradation of the drug occurred in acidic and neutral conditions. The drug was stable to dry heat. Resolution of drug and the degradation products formed under different stress studies were successfully achieved on a C-18 column utilizing water–methanol in the ratio of 85:15 and at the detection wavelength of 310 nm. The method was validated with respect to linearity, precision (including intermediate precision), accuracy, selectivity and specificity.

Controlling the Structure of a Trifluoroacylated Hydrazino Pharmaceutical Intermediate for Improved HPLC and GC Analyses

A process to generate an organofluorine pharmaceutical intermediate yields two main products, a bis‐ and mono‐trifluoroacylated analog of the hydrazino starting material. Analytical methods are discussed that characterize solid and solution intermediate mixtures, as well as monitor their formation. In addition, quantitative high performance liquid chromatography (HPLC) assay method development and validation is presented. However, due to solution stability issues, a diluent system that forcibly, yet controllably, degraded the bis‐trifluoroacylated product to the mono analog had to be employed. A more in depth discussion of the investigation into the appropriate diluent system is also presented.

Validated specific HPLC methods for determination of prazosin, terazosin and doxazosin in the presence of degradation products formed under ICH-recommended stress conditions

The present paper describes development of stability-indicating high-performance liquid chromatographic (HPLC) assay methods for three alpha-adrenergic-blocker drug substances, namely, prazosin, terazosin and doxazosin, in the presence of degradation products generated from forced decomposition studies. Resolution of drugs from degradation products was obtained using a reversed-phase C-18 column using water/acetonitrile/methanol/glacial acetic acid/diethylamine (25:35:40:1:0.017) as mobile phase for prazosin and terazosin and acetonitrile/water/glacial acetic acid/diethylamine (65:35:1:0.02) for doxazosin. The detection was done at 254 nm. The methods were validated with respect to linearity, precision, accuracy, specificity and robustness.

Determination of combined p-hydroxy benzoic acid preservatives in a liquid pharmaceutical formulation by HPLC

This paper describes a reversed-phase high performance liquid chromatographic (RP-HPLC) assay method for the determination of combined p-hydroxy benzoic acid (ethylparaben (EP), methylparaben (MP) and propylparaben (PP)) preservatives in a liquid pharmaceutical formulation. The chromatographic separation was achieved with potassium phosphate buffer (pH 7.05)-methanol (47.5:52.5, v/v) as mobile phase, a Spherisorb C 18 column ( 250 mm×4.6 mm) and UV detection at 254 nm. The analysis time was <8 min. The method was validated with respect to linearity, precision, accuracy, selectivity, specificity and ruggedness. The calibration curves showed good linearity over the concentration range of 2–140 μg/ml. The correlation coefficient were >0.9999 in each case. The relative standard deviation (R.S.D.) values for intra- and inter-day precision studies were <1%. The procedure describe here is simple, selective and is suitable for routine quality control analysis and stability tests.

Quantitation of tolmetin by high-performance liquid chromatography and method validation.

A high-performance liquid chromatographic (HPLC) assay method for assessing the degradation of tolmetin (TLM) is developed and validated under acidic, basic, and photoirradiated conditions. The HPLC method includes an Inertsil 5 ODS-3V column (250- x 4.6-mm i.d.), guard column of Inertsil 7 ODS-3V (50- x 4.6-mm i.d.), mobile phase of CH(3)OH-1% HOAc (64:36, v/v), and UV detection at 254 nm. The developed method satisfies the system suitability criteria, peak integrity, and resolution for the parent drug and its degradants. The established assay method exhibits good selectivity and specificity suitable for stability measurements. From the intra- and interday tests of six replicates, the coefficients of variation are between 0.20% and 1.77% for the former, and 0.12% and 3.40% for the latter. Recoveries are found to be 98.7-101.7%. TLM is determined to be more reactive when exposed to light and acidic conditions, yet TLM is stable in a basic medium. A kinetic study of the photodegradation of TLM shows that it follows an apparent first-order reaction in three alcoholic solvents.

Assay method for quality control and stability studies of a new CVS disorder agent (compound 93/478)

1-[4-(4-Fluorophenyl)-piperazine-1-yl]-3-(2-oxopyrrolidin-1-yl)-propane hydrochloride, ( I), (CDRI code No. 93/478) is a new potent anti-ischemic and anti-hypertensive agent, being developed at the Central Drug Research Institute (CDRI), Lucknow, India. A sensitive high performance liquid chromatographic assay method has been developed and validated for in process quality control and for stability studies. HPLC separation was achieved on a C 18 Purospher (Merck) column using a gradient of 0.02% tetra-methyl ammonium hydroxide (pH 7.5) and acetonitrile as mobile phase. The eluents were monitored by diode array detector at 240 and 290 nm. The lower limit of detection of I was 0.62 μg/ml, while the lower limit of quantitation was set to be 1.5 μg/ml. The calibration curves were linear in the range 1.5–62 μg/ml. Reproducibility of the method was determined by inter and intra assay variation, which were <10%.

Alternative Liquid Chromatographic Method for Determination of the Methoxyl and 2-Hydroxypropoxyl Content in Cellulose Ether Derivatives

An alternative liquid chromatographic (LC) method was developed and validated for the simultaneous determination of methoxyl and 2-hydroxypropoxyl substituents in hypromellose and hypromellose acetate succinate. The method uses the hydriodic acid cleavage reaction, catalyzed by adipic acid, of the substituted methoxyl and 2-hydroxypropoxyl groups, which are quantitatively converted to iodomethane and 2-iodopropane. The iodomethane and 2-iodopropane are extracted into xylene, the extract is diluted with methanol, and the analytes are separated and assayed by gradient elution using a reversed-phase C18 column. The method is selective and sensitive and has good linearity with values of 0.999 for R2 and 0.25% for the gamma-intercept bias from 1.39 to 5.55 mg/mL for iodomethane, and 0.999 for R2 and 0.52% for the gamma-intercept bias from 0.184 to 0.735 mg/mL for 2-iodopropane. The relative standard precisions for this LC method were found to be +/- 2.3% for determining methoxyl at the 23.1% (w/w) level, and +/- 3.5% for determining 2-hydroxypropoxyl at the 6.7% (w/w) level. Compared with the current gas chromatographic (GC) compendial (JPE) method, the LC assay method has equal or better precision. It was found that both the standard and sample solutions have limited stability (8 h) after preparation. This limited stability has not been reported previously in the literature and may have an impact on the reported accuracy/precision of the literature data for the GC method. The LC method was proven to be robust with respect to variation in derivatization time and temperature, flow rate, and column temperature. It is well suited for the quality control needed in today's fast-paced pharmaceutical laboratories.

Kinetics of kavain and its metabolites after oral application

Kavain metabolism in humans was the target of this current investigation. In the present study a high-performance liquid chromatographic (HPLC–DAD) assay method for the simultaneous determination of kavain and its main metabolites ( p-hydroxykavain, p-hydroxy-5,6-dehydrokavain and p-hydroxy-7,8-dihydrokavain) in serum and urine was developed and validated. The metabolites were mainly excreted in the form of their conjugates. All kavain metabolites were detectable in serum and urine, except for p-hydroxy-7,8-dihydrokavain, which was found in urine only. Confirmation of the results and identification of the metabolites were performed by LC–MS or LC–MS–MS. Kinetics of kavain and its metabolites in serum were investigated after administration of a single oral dose (800 mg kavain). Within 1 and 4 h after uptake, the serum concentrations ranged between 40 and 10 ng/ml for kavain, 300 and 125 ng/ml for p-hydroxykavain, 90 and 40 ng/ml for o-desmethyl-hydroxy-5,6-dehydrokavain, and 50 and 30 ng/ml for 5,6-dehydrokavain.

High-performance liquid chromatographic assay with fluorescence detection for the simultaneous measurement of carboxylate and lactone forms of irinotecan and three metabolites in human plasma

Irinotecan (CPT-11), a camptothecin analog, is metabolized to SN-38, an active topoisomerase I inhibitor, and inactive metabolites, including APC and SN-38 glucuronide (SN-38G). A high-performance liquid chromatographic assay method to simultaneously measure the lactone and carboxylate forms of CPT-11, SN-38, SN-38G, and APC in human plasma was developed. Chromatography was accomplished with a reversed-phase C 8 column and fluorescence detection. A gradient mobile phase system was used. The buffer for mobile phase A consisted of 0.75 M ammonium acetate, 5 m M tetrabutylammonium phosphate (pH 6.0), and acetonitrile (86:14, v/v). The buffer for mobile phase B was identical to mobile phase A with the exception of the concentration (50:50, v/v). Precipitation of plasma proteins was performed with cold methanol. The linear range of detection of the lactone and carboxylate forms of SN-38, SN-38G, and APC was 2–25 ng/ml, and 5–300 ng/ml for CPT-11. The limit of quantitation for the analytes ranged from 0.5 to 5 ng/ml. Analysis of patients’ plasma samples obtained before and after CPT-11 administration showed that the assay is suitable for measuring lactone and carboxylate forms of CPT-11, SN-38, SN-38G, and APC in clinical studies.

Validated liquid chromatography method for assay of tizanidine in drug substance and formulated products

A new isocratic stability indicating HPLC method for determination of tizanidine in drug substance and formulated products is described. Chromatographic separation of tizanidine from the related substances and degraded products was achieved with a Hypersil CN column ( 150 mm×5.0 mm, 5 μm) using a mobile phase comprising a mixture of an ion-pairing solution of heptanesulphonic acid sodium salt (HAS), methanol and acetonitrile (50:57:18 (v/v)) within 10 min. The flow-rate was 1.0 ml/min and detection was made at 227 nm. The method has good selectivity towards tizanidine, related substances and degraded products. Limits of quantitation for tizanidine and its synthetic intermediates were determined, ranging from 0.051 to 0.54 μg/ml. The linearity range was found to be 2–20 μg/ml ( r=0.9998, n=5). Mean recovery for tizanidine from the tablets was from 99.5 to 99.8%. Precision of the method was 1.0% ( n=9). The method can be used for routine analysis and the quality control of tizanidine drug substance and its formulated products.

Effect of inclusion complexation with cyclodextrins on photostability of nifedipine in solid state.

Nifedipine is a highly photosensitive drug that requires restricted protection from light during manufacturing, storage and handling of its dosage forms. Inclusion complexation of nifedipine with cyclodextrins (CDs) could be advantageous in protecting the drug against the effect of light. In this study, solid inclusion complexes of nifedipine with beta-cyclodextrin (beta-CD), hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and dimethyl-beta-cyclodextrin (DM-beta-CD) were prepared using the coprecipitation method. The obtained solid inclusion complexes have been confirmed by differential scanning calorimetry (DSC), X-ray diffraction and infrared spectroscopy (IR). The IR spectra indicated partial inclusion of nifedipine molecules into CD cavities through the dihydropyridine ring. Inclusion complexation was also associated with a dramatic enhancement of drug dissolution with magnitudes depended on the type of CD. The effect of exposure to fluorescent lamp and sunlight on the photodegradation of uncomplexed and complexed nifedipine was tested. Photodegradation of nifedipine was monitored using a high performance liquid chromatographic (HPLC) assay method. Inclusion complexation of nifedipine showed to retard drug photodegradation as indicated by degradation rate constant lowering with values depended on light source and type of complexing agent. This effect was the least with beta-CD compared with that of modified beta-CD. It was also interesting to notice that inclusion complexation of nifedipine offered much higher protection against the effect of fluorescent lamp than that of sunlight. The obtained results suggests that the design of solid dosage forms of nifedipine such as a fast dissolving nifedipine tablets is possible with the advantages of low required light protection.

A sensitive LC assay for the simultaneous determination of centbutindole and its metabolite in rat serum using fluorescence detection.

Centbutindole (+/-) 2-gamma-[(p-fluorobenzoyl)propyl]-1, 2, 3, 4, 6, 7, 12, 12a-octahydro-pyrazino (2', 1': 6, 1) pyrido [3, 4-b] indole (I), is a new neuroleptic agent developed by Central Drug Research Institute, India. In the present study, a high performance liquid chromatography (HPLC) assay method for the simultaneous assay for I and its metabolite (II) in rat serum was developed and validated. The present method requires only 1 ml of serum with detection levels similar to that reported earlier using 4 ml serum. This assay has been found to be more suited for pre-clinical as well as phase IV studies. Linearity was observed between 1.25 and 40 ng/ml for I and 0.625 and 20 ng/ml for II in rat serum. Recoveries were consistent for both the analytes over the concentration ranges studied. Variation in intra-and inter-batch accuracy and precision were within acceptable limits of +/-20% at lowest limit of quantitation, whereas at higher concentrations it was +/-15%. The assay method was employed for the study of the pharmacokinetics and metabolism of I in rats. The parent compound and its metabolites were quantitated in serum and could be monitored up to 24 h post dose.

High-performance liquid chromatographic assay with fluorescence detection for the determination of cephaeline and emetine in human plasma and urine

A high-performance liquid chromatographic assay method for the quantitation of ipecac alkaloids (cephaeline and emetine) in human plasma and urine is described. Human plasma or urine was extracted with diethylether under alkaline conditions following the addition of an internal standard. Concentrations of alkaloids and internal standard were determined by octadecylsilica chromatographic separation (Symmetry C 18 columns, plasma analysis; 15 cm×4.6 mm I.D., 5 μm particle size, urine analysis; 7.5 cm×4.6 mm I.D., 5 μm particle size). The mobile phase consisted of buffer (20 mmol/l 1-heptanesulfonic acid sodium salt, adjusted to pH 4.0 with acetic acid)–methanol (51:49, v/v). Eluate fluorescence was monitored at 285/316 nm. The lowest quantitation limits of cephaeline and emetine were 1 and 2.5 ng/ml, respectively, in plasma, and 5 ng/ml in urine. Intra- and inter-day relative standard deviations were below 15%. The assay is sensitive, specific and applicable to pharmacokinetic studies in humans.

Stability-indicating high-performance liquid chromatographic assay method and photostability of carprofen.

A rapid, sensitive, and accurate stability-indicating high-performance liquid chromatographic assay method for determining the degradation of carprofen (CPF) is developed and validated under acidic, basic, or photo-irradiated conditions. The analysis is monitored with a Cosmosil 5C18-AR column using a mobile phase of CH3CN-H2O-AcOH (50:49:1, v/v/v) at 260 nm. The developed method satisfies the system suitability criteria, peak integrity, and resolution among the parent drug and its degradation products. The results indicate that the established assay method shows good selectivity and specificity suitable for stability measurements of CPF. CPF is found to be more sensitive to exposure to light and in acidic conditions, but it is stable in a basic medium. The kinetic study of the photodegradation of CPF follows an apparent first-order reaction in a variety of solvents. The solvent effects on the rates of degradation are in the decreasing order of chloroform > dichloromethane > methanol > ethanol > 2-propanol, which is irrelevant to the dielectric constant epsilon. However, the hydrogen-donating ability of the solvents is essential to the photochemical decomposition of CPF. A plot of log k versus the Kirkwood function exhibits a linear relationship in aqueous ethanolic solutions, which implies that degradation proceeds via an ionic mechanism.

Fast isocratic high-performance liquid chromatographic assay method for the simultaneous determination of gabapentin and vigabatrin in human serum

Fast isocratic high-performance liquid chromatographic assay method for the simultaneous determination of gabapentin and vigabatrin in human serum

Validation of high-performance liquid chromatographic assay methods for the analysis of carboplatin in plasma ultrafiltrate

Validation of two HPLC assays for the quantitation of carboplatin in human plasma ultrafiltrate is described. Both assay methods employed a YMC ODS-AQ 3.9×150 mm (3 μm) column for the chromatographic separation. The first method utilized direct UV detection, the second method utilized UV detection following post-column derivatization with sodium bisulfite. Structural analogues of carboplatin were synthesized and used as internal standards for the assays. With direct UV detection, sample clean-up using solid-phase extraction on amino cartridges was required prior to injection, with extraction recoveries ranging from 80 to 90%. This extraction procedure was not necessary with the post-column reaction method, which employed a more selective analytical wavelength. Unfortunately, instability of the post-column reagent was a problem and led to greater variability in predicted concentration values. For standard curves, a weighted (1/ y 2) regression approach was used for plots of peak area or peak height ratio (carboplatin/internal standard) vs. carboplatin concentration. The limit of detection of both assays was 0.025 μg/ml and both were validated for carboplatin concentrations from 0.05 to 40 μg/ml. Accuracy and precision data were generated using three batches of validation samples, each batch consisting of a standard curve and five sets of quality control samples. Stability of carboplatin in blood, plasma, plasma ultrafiltrate, and reconstituted extracts was evaluated. The assay methods were employed for the pharmacokinetic analysis of blood samples drawn from a pediatric patient that received a 400 mg/m 2 dose of carboplatin.

Stability-indicating high-performance liquid chromatographic assay of buspirone HCl.

The United States Pharmacopoeia high-performance liquid chromatographic (HPLC) assay method of buspirone is not able to discriminate buspirone from its degradation products. The purpose of this work is to develop a sensitive, selective, and validated stability-indicating HPLC assay for the analysis of a buspirone hydrochloride in a bulk drug. Buspirone HCI and its potential impurities and degradation products are analyzed on an Ultrasphere C18 column heated to 40 degrees C using a gradient program that contains monobasic potassium phosphate buffer solution (pH 6.9) and acetonitrile-methanol mixture (13:17) of 35% for 5 minutes, then increased to 54% in 5.5 minutes. The samples are monitored using a photo-diode array detector and integrated at 244 and 210 nm. The stress testing of buspirone HCI shows that buspirone acid hydrochloride is the major degradation product. The developed method shows a separation of buspirone degradation product and its potential impurities in one run. The stability of buspirone HCI is studied under accelerated conditions in order to provide a rapid indication of differences that might result from a change in the manufacturing process or source of the sample. The forced degradation conditions include the effect of heat, moisture, light, acid-base hydrolysis, sonication, and oxidation. The compatibility of buspirone HCI with some pharmaceutical excipients is studied under stress conditions. The linear range of buspirone HCI is between 5 and 200 ng/microL with a limit of quantitation of 2.5 ng/microL. The intraassay percentage deviation is not more than 0.38%, and the day-to-day variation was not more than 0.80%. The selectivity, repeatability, linearity, range, accuracy, sample solution stability, ruggedness, and robustness show acceptable values.

Novel sensitive high-performance liquid chromatographic method for assay of coumarin 7-hydroxylation

In this paper, a novel HPLC-based method with fluorometric detection of coumarin 7-hydroxylase is presented. The described method provides a time-effective, more sensitive and specific alternative to the previously used spectrofluorometric assay. Using the developed method, metabolism of coumarin in 11 samples of human liver microsomes was evaluated and 1790±690 pmol/min/nmol cytochrome P450 (CYP) activity was found. Kinetic parameters and linearity of coumarin 7-hydroxylation were studied in a reconstituted system consisting of recombinant CYP2A6 expressed in Escherichia coli, rat NADPH-CYP reductase and usual components. It was found that a 3.5 to 30 min time of incubation is suitable for estimation of coumarin 7-hydroxylase activity. Observed K m and V max values in the CYP2A6 reconstituted system were 1.48±0.37 μ M and 3360±180 pmol product/min/nmol CYP, respectively.