Development and validation of a stability-indicating high performance liquid chromatographic assay for determination of cariprazine in bulk form and in drug product.

The aim of the present study is to develop a stability indicating high performance liquid chromatographic method for the determination of cariprazine in bulk substance and in drug product. The chromatographic separation was carried out using a Phenomenex Kinetex® C18 column (5µm, 250×4.6mm) and a mobile phase consisting of acetonitrile-potassium dihydrogen orthophosphate buffer (pH 4; 50mM) (30:70, v/v), at a flow rate of 1mlmin -1 and UV detection at 248nm. The column was maintained at 25°C and an injection volume of 20µL was used. Stress testing of cariprazine bulk substance and drug product was performed according to the International Conference on Harmonization (ICH) Q1A (R2) guideline. Various stress conditions were tested including acidic, alkaline and neutral hydrolysis, humidity, oxidation, dry heat and photolysis. A total of three degradation products (DPs) were formed. Among them two DPs were successfully characterized with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

Optimization of morphine extraction method for the assay of its urinary 3-glucuronideconjuguate by gas chromatography-mass spectrometry.

In the field of doping, a great interest is carried for the analysis of morphine, a powerful narcotic analgesic opiate which use is prohibited during competitions. In order to confirm the abnormal analytical result in our anti-doping laboratory, a sensitive and selective gas chromatography-mass spectrometry (GC-MS) method was performed for the quantification of urinary morphine. As sample preparation is a key step for the determination of drugs in biological samples, the aim of this work consists of the optimization of the urinary human sample pretreatment conditions before quantification by GC/MS. Enzymatic hydrolysis associated with liquid-liquid extraction constitute the major pre-treatment steps. Our study has first focused on the optimization of the extraction solvents then to enzymatic hydrolysis which morphine is released from its glucuronide conjugated form. Onboard premiums, a study involving the effect of "amount of enzyme", "incubation temperature" and "duration of hydrolysis" was conducted. This univariate study has enabled us to evaluate the influence of each of these operating variables on the area ratio of morphine to the internal standard (Amorphine/AIS) response and to set the experimental fields for each one of them. Based on these results, an experimental design was established using the Box-Behnken model to determine, by multivariate analysis, the optimal operating conditions maximizing the "Amophine/AIS" response. After validation, the analysis of response surface makes it possible to set the optimum operating conditions, which the ratio "Amorphine/AIS" is maximized. The retained conditions for enzymatic hydrolysis are 160µl of Escherichia coli glucuronidase enzyme during 6hours of incubation at a temperature of 36°C. The solvent mixture Methyl-t-Butyl Ether/isopropanol (4:1, v/v) was selected since it has improved morphine extraction from the urinary matrix allowing a gain of 50% when compared to that used in our routine laboratory. Our developed extraction method can be successfully applied for our forensic anti-doping analysis of morphin in human sample urine.

Development of a new stability indicating method for the simultaneous separation of voriconazole from its impurities along with sodium benzoate used as a preservative in a powder for oral suspension.

Voriconazole is a second-generation triazole derived from fluconazole, having an enhanced antifungal spectrum, compared with older triazoles. It is the drug of choice for treatment of invasive aspergillosis and many Scedosporium/Pseudallescheria Fusarium infections. Voriconazole is available in both intravenous and oral formulations. Since there is much interest in pharmaceutical quality control, separation of impurities from the main drug substances and accurate assay quantification, and since there is no reference or monograph until nowadays reported for the simultaneous separation of voriconazole from its specified and unspecified impurities along with sodium benzoate used as an antimicrobial preservative, our aim of this work is to develop a new simple, sensitive and stability indicating assay method allowing thus separation by high-performance liquid chromatography. The development of our method consisted in optimizing the following analytical parameters: nature and composition of the mobile phase, its pH, buffer concentration, nature of the stationary phase, column temperature and detection wavelength. After optimisation, separation was achieved on a stainless steel column NOVAPACK C18 (3.9mm×150mm; 4µm particle size) using a gradient mode with methanol, acetonitrile R and an aqueous solution acidified by acetic acid at 1% and adjusted to pH 2.77. The eluted compounds were monitored at 254nm. The flow rate was set at 1.0mL/min, the injection volume at 10µL, and the column oven temperature was maintained at 35°C. Under these conditions, separation was achieved with good resolution and symmetrical peaks' shape. The developed method was validated according to the International Conference on Harmonization (ICH) guidelines, and then it was successfully applied to establish inherent stability of the pharmaceutical formulation subjected to different ICH prescribed stress conditions. The developed method was proved to be simple, specific and precise. Hence, it can be considered as a method for stability study and for routine quality control analysis of voriconazole and sodium benzoate in a powder for oral suspension.

Development of a new liquid chromatographic method for the simultaneous separation of ciprofloxacin degradation products with 5-hydroxymethyl-furfural, impurity of D-glucose, in an intravenous solution for perfusion.

The place occupied by fluoroquinolones is very important clinically. Ciprofloxacin has been the most widely prescribed one which exhibits good activity against Pseudomonas aeruginosa and Acinetobacter. D-glucose is frequently used as an excipient in most formulations for perfusion solutions. Since there is much interest in pharmaceutical quality control of such formulations, separation of impurities from the main drug substances and accurate assay quantification, and since there is no reference or monograph until nowadays that has been reported for the simultaneous separation of ciprofloxacin degradation products along with 5-hydroxymethyl-furfural (5-HMF), impurity of D-glucose, classified as a high toxic substance, thus our aim of this work is to develop a new simple, sensitive and stability indicating method allowing this separation by high-performance liquid chromatography. We have started from the chromatographic conditions recommended by the British Pharmacopoeia, and by optimizing the nature of the stationary phase, the composition of the mobile phase and the injection volume. After optimisation, the retained chromatographic conditions have enabled the separation of all impurities with good resolution factor greater than 1.5 for each pair of peaks and with good symmetry peak shape. The developed method was validated according to the International Conference of Harmonisation (ICH) guidelines for specificity, detection and quantification limits, and then it was applied to stability study of the formulation subjected to different ICH prescribed stress conditions. The 5-HMF was checked to be the impurity issued from D-glucose hydrolysis by high temperature mainly after autoclaving of pharmaceuticals. The developed method was proved to be simple, specific with very low limit of quantification. Hence, it can be considered as a method for stability indicating and routine quality control analysis in pharmaceutical industries.

Development and validation of ultra-performance liquid chromatography method for the determination of meloxicam and its impurities in active pharmaceutical ingredients.

The main objective was to develop a suitable and rapid ultra-performance liquid chromatography (UPLC) method for the quantitative determination of meloxicam and its impurities. Starting from data in literature, we calculated the new parameters to translate a high performance liquid chromatography method used for the analysis of meloxicam with its major degradation products to UPLC method, and then we switched on many trials to optimize and improve its analytical performance. Chromatographic separation was achieved on ACQUITY UPLC HSS-T3 (2.1×100mm, 1.8µm). The eluted compounds were monitored at 260nm and 350nm. The flow rate was set at 0.4mL/min, injection volume at 0.8µl, and the column oven temperature was maintained at 45°C. The developed method was validated according to the International Conference on Harmonisation (ICH) guidelines for specificity, linearity, accuracy, precision, robustness, quantification limit, detection limit; and then applied to stability study of meloxicam subjected to different ICH prescribed stress conditions (hydrolysis, oxidation, heat and photolysis). The results show that the new UPLC method enables separation of meloxicam from its impurities in only 5min with a total mobile phase consumption of 1.8mL. All impurities get separated with good peak shapes and resolution factor greater than 2. The new method indicates stability and proved to be specific, precise accurate with linear correlation between concentrations and peak areas, allowing gain of more than six times analysis and more than twenty times solvent consumption, so in cost. Therefore, it can be beneficial for pharmaceutical industrial output.

Study of forced degradation behaviour of florfenicol by LC and LC-MS and development of a validated stability-indicating assay method.

A stability-indicating high performance liquid chromatography (HPLC) method was developed for the analysis of florfenicol in presence of its two available identified degradation products (thiamphenicol and chlorfenicol). The drug was subjected to different International Conference On Harmonisation (ICH) prescribed stress conditions (hydrolysis, oxidation and photolysis). The products formed under different stress conditions were investigated by liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS). The LC method involved a Knauwer Eurospher C18 thermostated column at 25°C; and ammonium acetate buffer 6.49mM (pH adjusted to 4.5)-methanol (70:30 v/v) as mobile phase. The flow rate and detection wavelength were 1ml/min and 225nm respectively. The drug showed instability under acidic, alkaline and photolytic stress conditions mainly in solution state form; however, it remains stable in solid state form and under oxidative stress conditions. The developed method was validated for linearity, precision, accuracy and specificity. The degradation products were characterized by LC-MS. Through the mass/ionization (m/z) values and fragmentation patterns, two principal degradation products listed in bibliography have been shown: the florfenicol amine and thiamphenicol. Based on the results, a more complete degradation pathway of the drug could be proposed.

Stress degradation study on sulfadimethoxine and development of a validated stability-indicating HPLC assay.

Sulfadimethoxine was subjected to different International Conference on Harmonisation prescribed stress conditions (hydrolysis, oxidation and photolysis). A stability-indicating high-performance liquid chromatography method was developed for analysis of the drug in the presence of its major degradation products. It involved a Knauer Eurospher C18 thermostated column at 25°C, and 9.57 mM phosphate buffer (pH adjusted to 2.0 with orthophosphoric acid)-acetonitrile (70:30 v/v) as mobile phase. The mobile phase flow rate and sample volume injected were 1.2 mL/min and 20 µL respectively. The selected wavelength for the determination was 248 nm. The method was validated for its linearity, precision, accuracy, specificity and selectivity, and then applied to the assay of sulfadimethoxine in pharmaceutical formulations. The results of the study show that sulfadimethoxine is highly sensitive to basic hydrolysis and oxidation. The mechanisms and schemas of hydrolytic, oxidative and photolytic degradation are also studied. The method developed, which separates all of the most degradation products, is simple, accurate, precise and specific. Thus, it can be applied to study the stability of veterinary preparations containing sulfadimethoxine.

Validation of a liquid chromatography method for the simultaneous determination of sulfadimethoxine and trimethoprim and application to a stability study.

A liquid chromatography method is described for the simultaneous determination of sulfadimetoxine and trimethoprim from a veterinary formulation at the proportion of 187 mg and 40 mg respectively in presence of some excipient. The solution was subjected to different International Conference On Harmonisation prescribed stress conditions (hydrolysis, oxidation and photolysis). A stability-indicating high-performance liquid chromatography method was developed for the analysis of active substances in presence of their major degradation products. It involved a Knauer Eurospher C18 thermostated column at 25 degrees C, and 9.57 mM phosphate buffer (pH adjusted to 2.0 with orthophosphoric acid)-acetonitrile (70:30 v/v) as mobile phase. The mobile phase flow rate and sample volume injected were 1.2 mL/min and 20 microL, respectively. The selected wavelength for the determination was 248 nm. The method was validated for linearity, precision, accuracy and specificity, and then applied to a stability study of sulfadimetoxine and trimethoprim in the veterinary solution packaged in high density polyethylene plastic bottles of 1 L and 100 mL thermosealed and no thermosealed and corked by a white cap, at both accelerated and long-term conditions required by the International Conference On Harmonisation. The method developed, which separates all of the most degradation products formed under variety of conditions, proved to be simple, accurate, precise and specific. The results of the stress degradation show that the solution is more sensitive to hydrolysis. The stability studies carried out on three batches of each presentation show that the finished product remains stable for six months.

Stability indicating LC method for the determination of pipamperone.

A high performance liquid chromatographic method for the determination of pipamperone in the presence of one related impurity and its degradation products is described. The method is based on the use of an amide functionalized bonded phase column (LC-ABZ+ Plus) and a mobile phase of acetonitrile-tetrahydrofuran-sodium phosphate monobasic (0.05 M, pH 6.5) (16:11:73, v/v/v). All peaks are eluted in <8 min. The method was demonstrated to be precise, accurate and specific. Degradation study showed that the drug is stable in acidic medium while it degrades under basic and oxidative conditions. The results indicated that the proposed method could be used in a stability assay.

Determination and degradation study of haloperidol by high performance liquid chromatography.

A specific, high performance liquid chromatographic method was developed for the assay of haloperidol, together with an adequate separation of its degradation products. The method is based on the use of an octadecylsilane stationary phase column under isocratic conditions. The mobile phase consisted of 50 mM sodium phosphate monobasic pH 2.5-acetonitrile-THF-TEA (63:34:3:0.1, v/v/v/v) adjusted with o-phosphoric acid to a pH of 2.5. The degradation was performed in hydrochloric acid, sodium hydroxide and hydrogen peroxide. The main degradation products were identified. Application of the assay of haloperidol in tablet formulations is presented.

LC determination and degradation study of droperidol.

A specific, high performance liquid chromatographic method for the determination of droperidol in the presence of its degradation products is described. The method is based on the use of an amide functionalized bonded phase column (LC-ABZ(+) Plus) and a simple mobile phase of methanol-sodium phosphate monobasic (0.05 M, pH 4.5) (40:60, v/v). It enables the resolution of eight compounds from the parent drug and from each other. The degradation was carried out in hydrochloric acid, sodium hydroxide and hydrogen peroxide. The main degradation products were identified. Application of the assay for a commercial preparation of droperidol for injection is presented.

Zero-crossing derivative spectrophotometry for the determination of haloperidol in presence of parabens.

First derivative spectrophotometry with a zero-crossing technique of measurement is used for the quantitative determination of haloperidol in the presence of methylparaben and propylparaben, which is added as antimicrobial preservatives in pharmaceuticals. This technique permits the quantification of haloperidol in the presence of parabens, with closely overlapping spectral bands, and without any separation step. Linear calibration graphs of first derivative values (at 255.2 nm for haloperidol) versus concentration (in the range 4.0-20.0 micrograms ml-1) were obtained with negligible intercepts. Relative standard deviation of 0.83% was obtained for intra-day precision and 1.86% for inter-day precision. The recovery of haloperidol in synthetic mixtures with parabens and pharmaceutical dosage forms is also reported.