HPLC‐ELSD analysis of spectinomycin dihydrochloride and its impurities

A simple, rapid and reliable reversed‐phase ion‐pair chromatography method by HPLC coupled to an evaporative light scattering detector (ELSD) has been developed to simultaneously determine chloride, spectinomycin and its related substances in a sample. The column was a TSKgel ODS‐100V. The mobile phase was ACN/aqueous solution of 15 mM ammonium acetate adjusted with TFA to pH 3.0 (2:98 v/v), in an isocratic mode. The drift tube temperature was set at 50°C and the nebulizing gas flow rate of air was 3.5 L/min for ELSD detection. Almost all of the reported degradation compounds of spectinomycin such as actinamine, actinospectinoic acid and biosynthesis intermediates such as dihydrospectinomycin diastereoisomers were baseline separated. MS was utilized for the identification of spectinomycin and its seven related substances. The method for the assay of spectinomycin was successfully validated with respect to accuracy, precision (RSD less than 2%), linearity (throughout the linear range 0.025–3 mg/mL, r=0.9993), sensitivity (LOD: 100 ng on column) and robustness. The experimental results demonstrated that the simultaneous determination of chloride, spectinomycin and related substances is feasible in a single run, which suggests applicability in routine assays.     

HPLC Analysis of Methoxamine in Rabbit Plasma and Pharmaceutical Formulations

Abstract

A rapid, selective and simple high performance liquid chromatographic (HPLC) assay for methoxamine HC1 has been developed. The analytical procedure involved the use of internal standardization method (1-norephedrine). It has been applied for the qualitative and quantitative determination of methoxamine HC1 in rabbit plasma and in pharmaceutical formulations using an adsorption column in an isocratic mode, with resulting relative standard deviations of 1.7% and 3.3%, respectively. The applicability of the assay procedure to pharmacokinetic studies was demonstrated. Detection limits were as low as 15ng for a 30 ul injection and the determination time was less than 6 minutes.     

A novel validated LC method for quantitation of lopinavir in bulk drug and pharmaceutical formulation in the presence of its potential impurities and degradation products

A simple isocratic liquid chromatographic method was developed for determination of lopinavir from its related impurities and assay for the first time. This method involves the use of a C(8) (Symmetry Shield RP8, 150 x 4.6 mm, 5 microm) column. The method was validated over the range of limit of quantitation (LOQ) to 120% of impurity specification limit and LOQ to 150% of working concentration for assay. The mobile phase consisted of a mixture of 50 mM of potassium phosphate buffer, acetonitrile and methanol in the ratio of 40:50:10. The flow rate was set at 1.0 mL/min with UV detection monitored at 210 nm. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The developed method was validated for linearity, range, precision, accuracy and specificity. This method was successfully applied for content determination of lopinavir in pharmaceutical formulations. The method can be conveniently used in a quality control laboratory for routine analysis for assay and related substances as well for the evaluation of stability samples of bulk drugs and pharmaceutical formulations.     

Development and validation of an RP-HPLC method for the estimation of adenosine and related purines in brain tissues of rats

A new, rapid and sensitive RP-HPLC method with UV spectrophotometric detection was developed and validated for the concomitant estimation of adenosine and related purines in rat brain tissue preparations. The HPLC system consisted of C-18 column with UV-photodiode-array detection ranging from 210 to 400 nm, facilitating the online confirmation of peak purity. The column temperature was maintained at 30 degrees C and the injection volume was 20 muL. Elution with an isocratic mobile phase consisting of water/methanol/acetonitrile (88:5:7 by volume) at a flow rate of 0.8 mL/min yielded sharp, utmost-resolved peaks of adenosine (Ade), inosine (Ino), hypoxanthine (Hypoxan) and adenine (Adn) within 10 min. The method was validated with respect to the linearity, accuracy, precision, sensitivity, selectivity and stability. The method was also employed to estimate the naturally occurring purines in discrete regions of rat brain. A new protocol developed for tissue preparation utilizing H(2)SO(4) and Tris buffer gave well-resolved peaks and high component recoveries (>96%) which eliminated the need of an internal standard. The results show that the method for the determination of Ade, Ino, Hypoxan and Adn by RP-HPLC described here has good linearity, accuracy, precision, sensitivity, selectivity and is simple and rapid to perform.     

Method development and validation study for quantitative determination of nifedipine and related substances by ultra‐high‐performance liquid chromatography

A novel stability‐indicating reversed phase ultra‐high performance liquid chromatography (UPLC) coupled photodiode array gradient method was developed for determination of the nifedipine and related compounds. Furthermore, based on the chromatographic conditions and forced degradation studies performed through the development of the related substances method a UPLC isocratic method was validated for the determination of the assay of this active substance. An Acquity Shield RP₁₈ (50 × 3.0 mm 1.7 µm) column was used for separation of nifedipine and its five potential impurities within 11 min, which is 5‐fold less than the official method. A mobile phase consisting of 10 mm ammonium formate (pH 4.5) and methanol, delivered at a flow rate 0.5 mL/min, was employed to achieve a minimum resolution of 2.0 for all consecutive pairs of compounds. The precision value expressed as percentage relative standard deviation for method repeatability and reproducibility was <5.0%. The recoveries for all the related compounds were in the range of 99–105.0%. Linearity was found to be acceptable over the concentration range of 0.25–1.5 µg/mL for nifedipine and its impurities. The limit of quantification for nifedipine was 0.05 µg/mL, which is much less than the European Pharmacopoeia method. Copyright © 2014 John Wiley & Sons, Ltd.     

New gradient high-performance liquid chromatography method for determination of donepezil hydrochloride assay and impurities content in oral pharmaceutical formulation

A new gradient HPLC method has been developed and validated for the determination of both assay and related substances of donepezil hydrochloride in oral pharmaceutical formulation. Different kinds of columns and gradient elution programs were tested in order to achieve satisfactory separation between the active substance, four impurities and an interfering excipient used in the formulation. The best results were obtained using an Uptisphere ODB C-18 column 250 mm x 4.6 mm, 5 microm, UV detection at 270 nm and a gradient elution of phosphate buffer (0.005 M, pH 3.67) and methanol as the mobile phase. The method was validated with respect to linearity, precision, accuracy, specificity and robustness. It was also found to be stability indicating, and therefore suitable for the routine analysis of donepezil hydrochloride and related substances in the pharmaceutical formulation.     

A Validated Stability Indicating RP-LC Method for Nitazoxanide, a New Antiparasitic Compound

The present paper describes stability indicating reverse phase high-performance liquid chromatography (RP-HPLC) assay method for nitazoxanide in bulk drugs. The developed method is also applicable for the related substances determination in bulk drug. The drug substance was subjected to stress conditions of hydrolysis, photolysis and thermal degradation. The considerable degradation of nitazoxanide was observed under base and peroxide hydrolysis. The drug was found to be stable in other stress conditions attempted. The chromatographic separation of the drug was achieved on reversed-phase C-18 column. Eluents were monitored on photo-diode array detector at a wavelength of 240 nm. The mobile phase was aqueous 0.005 M tetra butyl ammonium hydrogen sulphate and acetonitrile (45:55, v/v). In the developed HPLC method, resolution between nitazoxanide and its potential impurities, namely Imp-A (5-nitro-1,3-thiazol-2-amine), Imp-B (N-(5-nitro-1,3-thiazol-2-yl) acetamide) and Imp-C (2-{[(5-nitro-1,3-thiazol-2-yl) amino] carbonyl} phenyl 2-(acetyloxy) benzoate) was found greater than three. The developed RP-HPLC method was validated with respect to response function, accuracy, precision, specificity, stability of analytical solutions and robustness. Also to determine related substances and assay determination of nitazoxanide that can be used to evaluate the quality of regular production samples. The developed method can also be conveniently used for the assay determination of nitazoxanide in pharmaceutical formulations.     

Development and validation of high-performance liquid chromatography method for analysis of β-CCT and its related substances

Compound 2β-carbomethoxy-3β-(4-chlorophenyl)tropane (β-CCT) is a key intermediate for the synthesis of some clinical dopamine transporter (DAT) imaging agents. Potential impurities from synthesis process of β-CCT and degradation during storage might have detrimental effect on the final imaging agents. Thus, it is necessary to guarantee the quality of β-CCT. In this study, a rapid, sensitive and accurate high-performance liquid chromatography (HPLC) method was developed and validated for the analysis of β-CCT and its related substances. The chromatographic separation was achieved on a reverse-phase phenomenex? Gemini C18 column with an isocratic mobile phase consisted of methanol, water and TFA (30:70:0.1 v/v/v). The flow rate was 1.0 mL/min at 30 °C and samples were monitored at 220 nm. The method was validated concerning system suitability, linearity, accuracy, precision, specificity, robustness and stability. The limit of detection (LOD) and the limit of quantification (LOQ) of β-CCT were 0.5 and 1.5 μg/mL, respectively. The linearity range of β-CCT was 1.5–450 μg/mL with a good linear correlation coefficient (R² = 0.9999) between the peak response and concentration. Specificity investigation through forced degradation experiments displayed that β-CCT was stable in acidic, thermal and photolytic degradation conditions, but significantly unstable in alkaline and oxidative conditions. With the developed chromatographic method, possible impurity α-CCT from synthetic process and potential degradation products could be well separated from β-CCT. Good recovery and precision were manifested in the assay method. These results indicated that the present method would be suitable for not only the quality assurance of β-CCT in regular production sample assays but also the monitoring and determination of its related substances.     

Photochemical properties of simvastatin and lovastatin induced by radiation

HPLC and UV spectrophotometry were developed and validated for quantitative determination of two antihiperlipoproteinemia drugs, lovastatin and simvastatin. Analytical performance parameters such as linearity, precision, specificity, limit of detection (LOD) and limit of quantification (LOQ) were determined according to the ICH Q1B guidelines. Chromatography was carried out by isocratic technique on a reversed-phase C-18 column. The UV spectroscopy determinations were performed at 238 nm. The linearity of the calibration curves in the desired concentration range was good (r(2)>0.999) for both HPLC and UV methods. The relative standard deviation (RSD) for these methods was <5%. Moreover, the precision obtained with HPLC correlated well with the UV results. The methods proposed are highly sensitive and precise. Other methods used for assessment of the photostability of the substances studied were electron paramagnetic resonance (EPR) and differential scanning calorimetry (DSC).     

Development and Validation of an HPLC Method for the Determination of Dexamethasone, Dexamethasone Sodium Phosphate and Chloramphenicol in Presence of Each Other in Pharmaceutical Preparations

An HPLC method for the determination of dexamethasone, dexamethasone sodium phosphate and chloramphenicol in presence of each other in pharmaceutical preparations has been developed using a Shim-Pack CLC-ODS column (6.0 × 150 mm²). These analytes were separated under isocratic conditions. Various chromatographic parameters including linearity, precision and accuracy have been evaluated. The method was found to be suitable for analysis of these drug substances in presence of each other. The run time was less than 15 min. This method is suitable for application to various dosage forms.     

Development and validation of a reverse-phase liquid chromatographic method for assay and related substances of abacavir sulfate

A simple isocratic liquid chromatographic method was developed for the determination of abacavir from its related substances and assay for the first time. This method involves the usage of C18 (Intertsil octadecyl silane-3V, 150 mm x 4.6 mm, 5 microm) column. The method was validated over the range of 0.002-0.1 mg/mL for chloro impurity, 0.005-0.1 mg/mL for amino impurity and pyrimidine impurity, and 0.005-0.2 mg/mL for abacavir. The mobile phase consists of a mixture of 10 mM ammonium acetate buffer and ACN in the ratio of 90:10. The flow rate was set at 1.0 mL/min with UV detection monitored at 214 nm. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis, and thermal degradation. The developed method was validated for linearity, range, precision, accuracy, and specificity. This method can be conveniently used in a quality control laboratory for routine analysis of both related substances and assay.     

Enantioselective determination of R‐(−)‐manidipine and S‐(+)‐manidipine in human plasma by a sensitive and selective chiral LC–MS/MS assay

A sensitive and selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay method has been developed and validated for the enantioselective determination of manidipine in human plasma using isotope‐labeled compounds as internal standards. After solid‐phase extraction, R ‐(−)‐manidipine and S ‐(+)‐manidipine were chromatographed on a Chiralpack IC‐3 C₁₈ column using a isocratic mobile phase composed of 2 mm ammonium bicarbonate and acetonitrile (15:85, v /v). The precursor ion to product ion transitions for the enantiomers and internal standards were monitored in the multiple reaction monitoring and positive ionization mode using an API‐4000 mass spectrometer. The method was linear over the concentration range of 0.05–10.2 ng/mL for both enantiomers. The precision and accuracy results over five concentration levels in five different batches were well within the acceptance limits. The mean extraction recovery was >80% for both enantiomers. A variety of stability tests were executed in plasma and in neat samples, which complies with the FDA guidelines. After complete validation, the method was successfully applied to a pharmacokinetic study of a manidipine 20 mg oral dose in 10 healthy South India subjects under fasting conditions. The assay reproducibility is shown through incurred samples reanalysis of 20 subject plasma samples.     

Determination of mitragynine in plasma with solid-phase extraction and rapid HPLC–UV analysis, and its application to a pharmacokinetic study in rat

A new solid phase extraction method for rapid high performance liquid chromatography–UV determination of mitragynine in plasma has been developed. Optimal separation was achieved with an isocratic mobile phase consisting of acetonitrile–ammonium acetate buffer, 50 mM at pH 5.0 (50:50, v/v). The method had limits of detection and quantification of 0.025 and 0.050 μg/mL, respectively. The method was accurate and precise for the quantitative analysis of mitragynine in human and rat plasma with within-day and between-day accuracies between 84.0 and 109.6%, and their precision values were between 1.7 and 16.8%. Additional advantages over known methods are related to the solid phase extraction technique for sample preparation which yields a clean chromatogram, a short total analysis time, requires a smaller amount of plasma samples and has good assay sensitivity for bioanalytical application. The method was successfully applied in pharmacokinetic and stability studies of mitragynine. In the present study, mitragynine was found to be fairly stable during storage and sample preparation. The present study showed for the first time the detailed pharmacokinetic profiles of mitragynine. Following intravenous administration, mitragynine demonstrated a biphasic elimination from plasma. Oral absorption of the drug was slow, prolonged and was incomplete, with a calculated absolute oral bioavailability value of 3.03%. The variations observed in previous pharmacokinetic studies after oral administration of mitragynine could be attributed to its poor bioavailability rather than to the differences in assay method, metabolic saturation or mitragynine dose.     

Determination of piquindone in canine plasma and urine by high-performance liquid chromatography

This report describes a rapid, sensitive and selective method for the determination of piquindone in canine plasma and piquindone and the N-demethyl metabolite of piquindone in canine urine, utilizing normal-phase high-performance liquid chromatography (HPLC) with isocratic elution at ambient temperature and monitoring the ultraviolet absorbance of the eluent at 254 nm. The trimethyl analogue of piquindone is used as the internal standard in the HPLC assay of plasma. The assay was applied to the measurement of concentrations of piquindone in the plasma and urine of a dog following single intravenous and oral administration of 5 mg/kg doses of piquindone hydrochloride dihydrate.     

Determination of nivalenol and deoxynivalenol in wheat using liquid chromatography-mass spectrometry with negative ion atmospheric pressure chemical ionisation.

A new, rapid and sensitive method has been developed for the determination of nivalenol (NIV) and deoxynivalenol (DON) by using HPLC in combination with an atmospheric pressure chemical ionization (APCI)-interface and a single quadrupole mass spectrometer. Different LC and MS parameters have been optimized prior to this in order to obtain better results and sensitivity. The effect of nebulizing temperature on the sensitivity and fragmentation of NIV and DON in an APCI interface was investigated. Also, the influence of the cone voltage on the fragmentation pattern was studied, which was shown to have a tremendous effect. Furthermore, the effect of modifiers such as ammonium acetate, acetic acid and ammonia on the ionisation yield of the above substances have been investigated. The extraction was carried out using acetonitrile-water. A two step purification was then applied on two different Mycosep clean up columns. We have used a modified, rapid and isocratic HPLC method combined with a negative ion APCI-MS for the separation and quantitative determination of NIV and DON in wheat extract. An RP C18 column was used for the separation of selected compounds in wheat extract with water-acetonitrile-methanol (82:9:9, v/v/v) at a flow-rate of 1 ml/min without a split. Calibration curves show good linearity and reproducibility. The detection limit and precision were determined for NIV and DON. Both compounds could be detected down to microg/kg level in wheat using selected ion monitoring of the [M-H]- ions and the main fragments.     

Determination of 8[(3S)-3-amino-1-pyrrolidinyl]-1-cyclopropyl-7-fluoro-9-methyl-4-oxo-4H-quinolizine-3-carboxylic acid hydrochloride and related substances by high performance liquid chromatography

Summary The purity of the investigational antibacterial Abbott-086719.1 (I) which is 8[(3S)-3-amino-1-pyrrolidinyl]-1-cyclopropyl-7-fluoro-9-methyl-4-oxo-4H-quinolizine-3-carboxylic acid hydrochloride is determined using gradient elution HPLC. The chromatographic separation was optimized and the chromatographic parameters critical to separation are discussed in detail. Most acceptable separations were achieved using an Alltima C18 column (5 m) measuring 4.6 mm I.D.×15 cm with 0.01M–0.03M citrate eluents which were modified with acetonitrile or with mixtures of acetonitrile and methanol. Related substances in I were determinable to 0.05%. Repeatability (RSD values) for determining related substances at levels of 0.50 to 0.06% ranged from ±1.9 to ±10%. Determinations of I in 5% dextrose in water and in hydroxypropyl methylcellulose were made using the same column and a simple isocratic system. The determination of I was stability indicating with precision (RSD values) of ±0.5% to ±2.0% and good agreement with theory for formulations containing I at 1.0 to 95 mg/mL concentrations. Recoveries of I from the vehicles were quantitative and linearity of the detector response of I was demonstrated to at least 0.10 mg/mL.     

Determination and confirmation of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment using high-performance liquid chromatography/tandem mass spectrometry

An accurate, reliable, and reproducible analytical method using HPLC/MS/MS for the determination of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment (CP-60,300) was developed and validated. Briefly, the method involved an initial acid treatment of intact tissues, which yielded the common fragment (CP-60,300). A portion of the acid hydrolyzate was purified by SPE using a strong cation exchange cartridge. Evaporation of the purified extract was followed by reconstitution in aqueous buffer and analysis by HPLC/MS/MS under isocratic conditions. The developed method provided acceptable sensitivity for determinative surveillance of tulathromycin in porcine kidney and bovine liver with an LOQ of 7.50 and 2.75 microg/g, respectively. The overall recovery and precision of 45 determinations of each tissue were 97.8% (5.3%) for porcine kidney and 96.9% (7.9%) for bovine liver. Accuracy, precision, linearity, specificity, and ruggedness were demonstrated. An HPLC/MS/MS method was also developed for use in these tissues as a confirmatory assay following modifications to the MS detection parameters. The confirmatory method demonstrated acceptable sensitivity for confirmatory evaluation of tulathromycin in porcine kidney and bovine liver at tolerances of 15 and 5.5 microg/g, respectively.     

Development and comparison of HPLC and MEKC methods for the analysis of cyclic sulfur mustard degradation products

In this study, novel, fast, and simple methods based on RP‐HPLC and MEKC with DAD are developed and validated for the qualitative and quantitative determination of five cyclic sulfur mustard (HD) degradation products (1,4‐thioxane, 1,3‐dithiolane, 1,4‐dithiane, 1,2,5‐trithiepane, and 1,4,5‐oxadithiepane) in water samples. The HPLC method employs a C18 column and an isocratic water‐ACN (55:45, v/v) mobile phase. This method enables separation of all five cyclic compounds within 8 min. With the CE method, the baseline separation of five compounds was achieved in less than 11 min by applying a simple BGE composed of a 10 mM borate buffer and 90 mM SDS (pH 9.15). Both methods showed good linear correlation (R ² > 0.9904). The detection limits were in the range of 0.08–0.1 μM for the HPLC method and 10–20 μM for MEKC. The precision tests resulted in RSDs for migration times and peak areas less than 0.9 and 5.5%, respectively, for the HPLC method, and less than 1.1 and 7.7% for the MEKC method, respectively. The developed methods were successfully applied to the analysis of five cyclic HD degradation products in water samples. With the HPLC method, the LODs were lowered using the SPE for sample purification and concentration.     

Stability indicating RP-HPLC method development and validation for oseltamivir API

The present study describes the development and subsequent validation of a stability indicating reverse-phase HPLC (RP-HPLC) method for the analysis of oseltamivir active pharmaceutical ingredient (API). The proposed RP-HPLC method utilizes Kromasil C(18), 5 microm, 250 mm x 4.6 mm i.d. column (at ambient temperature), gradient run (using acetonitrile and triethylamine as mobile phase), effluent flow rate (1.0 ml/min), and UV detection at 215 nm for analysis of oseltamivir. The described method was linear over the range of 70-130 microg/ml (r(2)=0.999). The precision, ruggedness and robustness values were also within the prescribed limits (<1% for system precision and <2% for other parameters). Oseltamivir was exposed to acidic, basic, oxidative and thermal stress conditions, and the stressed samples were analyzed by the proposed method. Chromatographic peak purity results indicated the absence of co-eluting peaks with the main peak of oseltamivir, which demonstrated the specificity of assay method for estimation of oseltamivir in presence of degradation products. The proposed method can be used for routine analysis of oseltamivir in quality control laboratories.     

A selective high-performance liquid chromatography method for the determination of reboxetine in bulk drug and tablets

Reboxetine is used as a selective noradrenaline reuptake inhibitor for the treatment of major depressive disorders. It is effective in the treatment of severe depression and safer to use than traditional tricyclic antidepressants. In this study, a novel, simple, and rapid stability-indicating high-performance liquid chromatography (HPLC) method for reboxetine methansulfonate was successfully developed and validated for the assay of tablets. The method was used to quantify reboxetine in tablets; it employed a C18 column (150 x 4.6 mm id) with an isocratic mobile phase consisting of methanol-phosphate buffer (pH 7, 0.02 M; 55 + 45, v/v) at a flow rate of 1.0 mL/min. Reboxetine was detected by an ultraviolet detector at 277 nm. The retention time of reboxetine was about 4.5 min. The developed HPLC method was validated with respect to linearity, precision, sensitivity, accuracy, and selectivity. The method was linear over the concentration range 1-50 microg/mL (r = 0.9999). The limits of detection and the quantitation of reboxetine were 0.1 and 0.3 microg/mL, respectively. The relative standard deviation values for intraday and interday precision were 0.78-1.01 and 1.08-1.37%, respectively. Selectivity was validated by subjecting a stock solution of reboxetine to neutral, acid, and alkali hydrolysis, as well as oxidation, dry heat treatment, and photodegradation. The peaks of the degradation products did not interfere with the peak of reboxetine. The results indicated that the proposed method could be used in a stability assay. The proposed method was successfully applied to the determination of reboxetine in tablets. Excipients present in the tablets did not interfere with the analysis.