A Chemometrical Approach to Optimization and Validation of an HPLC Assay for Rizatriptan and its Impurities in Tablets

Abstract

An isocratic reversed‐phase high‐performance liquid chromatographic method was developed and validated for the analysis of a novel antimigraine drug, rizatriptan benzoate, in a dosage form along with its two impurities, L‐749.019 and L‐783.540. The method used a C₁₈ XTerra? (150×3.9 mm), 5 µm column. The mobile phase consisted of a mixture of methanol, TEA (1%) and 10 mM KH₂PO₄ (5:9.5:85.5 v/v) at a flow rate of 1.2 ml min^?1 (pH of the water phase was adjusted to 5.5 with 85% orthophosphoric acid). Column temperature was 20°C and the detection was performed at 225 nm. The central composite design technique and the response surface method were used in the robustness test considerations. The method was applied satisfactorily to the analysis of commercial rizatriptan formulation.     

Development and Validation of an RP-HPLC Method for Determination of Valganciclovir in Human Serum and Tablets

This paper describes the validation of an isocratic high-performance liquid chromatographic method for the assay of valganciclovir in raw materials, tablets and human serum samples. Valganciclovir and fluvastatin (internal standard) were well separated using a reversed phase column and a mobile phase consisting of a mixture of acetonitrile:methanol:KH₂PO₄ (0.02 M) (40:20:40; v/v/v) (at pH 5.0). The mobile phase was pumped at 1.0 mL min⁻¹ flow rate and valganciclovir was detected by diode-array detection at 255 nm. The retention times for valganciclovir and fluvastatin were 3.41 and 5.60 min, respectively. A linear response (r > 0.999) was observed in the range of 10–30,000 ng mL⁻¹ in mobile phase and serum. The limit of detection and limit of quantification were found as 2.95 and 9.82 ng mL⁻¹ in mobile phase and 1.73 and 5.77 ng mL⁻¹ in human serum samples, respectively. Validation parameters as precision, accuracy, selectivity, reproducibility and system suitability tests were also determined. The method can be used for valganciclovir assay of tablets and human serum samples as the method separates valganciclovir from tablet excipients and endogenous substances.

     

UPLC Method for Determination of Moxonidine and Its Degradation Products in Active Pharmaceutical Ingredient and Pharmaceutical Dosage Form

A simple, rapid, isocratic, stability-indicating reverse phase ultra-performance liquid chromatographic (RP-UPLC) method was developed and validated for the routine analysis of moxonidine in the presence of its degradation products in active pharmaceutical ingredient and pharmaceutical dosage forms. Forced degradation studies were performed according to the guidance of International Conference for Harmonization and were used to evaluate moxonidine intrinsic stability. The drug was subjected to acid, neutral and base hydrolysis as well as to oxidative, thermal and photolytic decomposition in both solution and solid state. The drug appeared to be unstable towards acid and base hydrolysis. To achieve desirable conditions for UPLC analysis, the method development was done with the assistance of experimental design and multivariate optimization methodology by means of Derringer’s desirability function. Stress samples were analyzed according to the experimental plan for fractional factorial screening design and Box-Behnken optimization design. The chromatographic separation was achieved on a C₁₈ Hypersil Gold aq. column (100 mm × 2.1 mm, 1.9 μm) with the mobile phase consisting of methanol–ammonium acetate buffer (10 mM, pH 3.43) mixture (0.9:99.1, v/v) pumped at a flow rate of 870 μL min^?1 and detection wavelength of 255 nm. The UPLC–MS and UPLC–MS/MS analyses were further used to characterize the found degradation products. The validation of the proposed method was also performed considering selectivity, linearity, accuracy, precision, limit of detection and limit of quantification, and the results indicated that the method fulfilled all required criteria. The method was successfully applied to the analysis of commercial tablets.     

An Improved HPLC Method for Simultaneous Analysis of Losartan Potassium and Hydrochlorothiazide with the Aid of a Chemometric Protocol

An experimental design method was used for fast, simple, and accurate high-performance-pressure liquid chromatograpy (HPLC) determination of losartan and hydrochlorothiazide in combined dosage forms. This method avoids the disadvantages of the traditional analytical approach, which is time-consuming, involves a large number of runs, and does not allow the determination of multiple interacting parameters. On the basis of preliminary experiments, three independent variables (methanol content, pH value of the mobile phase, and flow rate) were selected as input, and as dependent variables, five responses (retention time of hydrochlorothiazide, retention time of losartan, asymmetry of hydrochlorothiazide peak, asymmetry of losartan peak, and resolution) were chosen. A full 2³ factorial design was used to determine which factors had an effect on the studied response. Afterwards, experimental design was used to optimize these influencing parameters in the previously selected experimental domain. After optimizing the experimental conditions, a separation was conducted on a Zorbax C₈ (150 mm × 4.6 mm, 5 μm particle size) column with a mobile phase consisting of methanol–acetonitrile–acetate buffer 45:20:35 v/v/v, pH 4.8 with flow rate of 0.82 mL min^?1 and column temperature of 25 °C. The developed method was successfully applied to simultaneous separation of these active drug compounds in their commercial pharmaceutical dosage forms.     

Multiobjective Optimization Approach in Evaluation of Chromatographic Behaviour of Zolpidem Tartrate and Its Degradation Products

Multiresponse optimization methodology in combination with experimental design was employed as a powerful technique for simultaneous optimization of input variables significant for evaluation of chromatographic behaviour of zolpidem tartrate, zolpacid, oxozolpidem, zolpyridine and zolpaldehyde towards various responses. In the first stage of the investigation fractional factorial design was used to decrease the number of variables that should be studied in detail. Among examined variables, pH of the mobile phase, percentage of organic modifier and buffer concentration showed to be statistically important and were consequently optimized with central composite design and Derringer??s desirability function. Four responses were considered, the retention factors of zolpacid and zolpaldehyde (the first and last peak) and the resolutions between critical peaks. Optimal conditions included Luna C₁₈(2) analytical column (250?mm?x?4.6?mm, 5???m particle size), mobile phase consisted of methanol?C10?mM ammonium acetate (68.4:31.6, v/v, pH 5.4) and column temperature of 35???C. The flow rate of the mobile phase was 1?mL?min^?1 and the detection was performed at 254?nm. At the end, the method was successfully validated in accordance with ICH guideline and subsequently applied to the analysis of commercially available zolpidem tartrate tablets.     

RP-LC and HPTLC Methods for the Determination of Olmesartan Medoxomil and Hydrochlorothiazide in Combined Tablet Dosage Forms

Two new, rapid, precise, accurate and specific chromatographic methods were described for the simultaneous determination of olmesartan medoxomil and hydrochlorothiazide in combined tablet dosage forms. The first method was based on reversed phase liquid chromatography using an Eurosphere 100 RP C18 column (250 × 4.6 mm ID, 5 μm). The mobile phase was methanol–0.05% o-phosphoric acid (60:40 v/v) at a flow rate of 1.0 mL min^?1. Commercially available tablets and laboratory mixtures containing both drugs were assayed and detected using a UV detector at 270 nm. The second method involved silica gel 60 F₂₅₄ high performance thin layer chromatography and densitometric detection at 254 nm using acetonitrile–ethyl acetate–glacial acid (7:3:0.4 v/v/v) as the mobile phase. Calibration curves ranged between 200–600 and 125–375 ng spot^?1 for olmesartan and hydrochlorothiazide, respectively.     

Development and Validation of Liquid Chromatography Method for the Separation of Valdecoxib and its SC‐77852 Impurity

Abstract

A reversed‐phase liquid chromatography method has been developed for the separation of valdecoxib and impurity SC‐77852. The best results were achieved using a mobile phase—methanol: 1% water solution TEA (52∶48 v/v), pH 7.35 (adjusted with 85% orthophosphoric acid), column temperature 24°C. Separation was carried out on XTerra? RP₁₈ (150 mm×4,6 mm), particle size 5 µm, flow rate 1 ml/min, using detection on 220 nm. The method was statistically validated for its selectivity, linearity, precision (repeatability), and robustness. Quantitation and detection limits were determined for both valdecoxib and SC‐77852. Method robustness was further evaluated by performing full factorial design experiment. Validated method was used for assay of valdecoxib and SC‐77852 in Bextra^® film‐coated tablets.     

Development and Validation of Stability Indicating LC Method to Quantify Captopril in Tablets of Controlled Release

An accurate, simple, reproducible, and sensitive liquid chromatographic method was developed and validated for the captopril determination in controlled release tablets. The analyses were performed at room temperature on a reversed-phase Phenomenex Luna C₁₈ column (250 mm × 4.6 mm). The mobile phase was composed of water:methanol (45:55; v/v) pH 2.5, and it was eluted isocratically at a 1.0 mL min⁻¹ flow rate. The method was validated in terms of specificity, linearity, quantification limit, detection limit, accuracy, precision and robustness. The response was linear in the range 0.3–1.5 mg mL⁻¹ (r ² = 0.9983). The relative standard deviation values for inter-and intra-day precision were 0.77% and 0.50%, respectively. Recoveries ranged between 97.7 and 99.1%. The method was successfully applied for the determination of captopril in the developed formulations.

     

RP-LC and HPTLC Methods for the Determination of Olmesartan Medoxomil and Hydrochlorothiazide in Combined Tablet Dosage Forms

Two new, rapid, precise, accurate and specific chromatographic methods were described for the simultaneous determination of olmesartan medoxomil and hydrochlorothiazide in combined tablet dosage forms. The first method was based on reversed phase liquid chromatography using an Eurosphere 100 RP C18 column (250 × 4.6 mm ID, 5 μm). The mobile phase was methanol–0.05% o-phosphoric acid (60:40 v/v) at a flow rate of 1.0 mL min⁻¹. Commercially available tablets and laboratory mixtures containing both drugs were assayed and detected using a UV detector at 270 nm. The second method involved silica gel 60 F₂₅₄ high performance thin layer chromatography and densitometric detection at 254 nm using acetonitrile–ethyl acetate–glacial acid (7:3:0.4 v/v/v) as the mobile phase. Calibration curves ranged between 200–600 and 125–375 ng spot⁻¹ for olmesartan and hydrochlorothiazide, respectively.

     

应用质量源于设计的理念优化大鼠血浆中大黄蒽醌的分析方法

应用质量源于设计理念建立一种高效液相色谱-荧光检测法(HPLC-FLD)用于测定大鼠血浆中5种大黄蒽醌。用Plackett-Burman设计考察流动相中甲醇含量、pH值、流速、柱温和进样体积对色谱峰的分离度、理论塔板数、最末洗脱峰的保留时间和拖尾因子的影响,结果显示流动相中甲醇含量、流速和柱温对色谱系统的影响显著(p<0.05)。继而采用Box-Behnken设计结合响应面法考察上述三因素对分离度、保留时间和理论塔板数的影响。用Derringer渴求函数评价了响应值的综合作用。得出最优色谱条件为:以甲醇-0.1%(v/v)磷酸水溶液(81.4:18.6, v/v)为流动相等度洗脱,流速1.1 mL/min,柱温31℃,荧光检测激发波长为440 nm,发射波长为540 nm。建立的模型显示良好的预测性。结果表明:质量源于设计的理念可有效地应用于优化高效液相色谱分析方法。     

Orthogonal Design-Directed Optimization of an LC Method for Fingerprinting Mai-Luo-Ning Injection, and Validation of the Method

A novel hierarchical chromatographic response function (HCRF)-directed orthogonal design procedure has been used for optimization of an high-performance liquid chromatography method for fingerprinting Mai-Luo-Ning (MLN) injection. The method was then successfully validated. Five major controllable chromatographic conditions at four levels were included in the orthogonal design. A total of 16 chromatographic runs resulted in the optimum chromatographic conditions-a 250 × 4.6 mm i.d., 4-μm particle, C₁₈ column, a mixture of methanol and 0.025% aqueous formic acid in water as mobile phase, flow rate 0.8 mL min^?1, column temperature 35 °C, and detection wavelength 240 nm. The mobile phase gradient was then further optimized step by step by observation of the chromatographic profiles obtained. Fingerprints of MLN injection and its constituent single herb injections were separately acquired by use of the optimized method. Attribution of the 18 largest peaks observed in the MLN fingerprint indicated that Flos Lonicerae was the main ingredient. Validation of the method for precision, repeatability, and stability proved it was highly reproducible. This chromatography fingerprint method could be very useful for quality control of MLN injection. The original HCRF-directed orthogonal design approach proposed should be generally useful for developing chromatographic fingerprinting methods.     

Crossed D-Optimal Experimental Design Methodology for the Chromatographic Separation of Antihypertensive Drugs

An efficient, isocratic, reversed-phase high performance liquid chromatography method was developed, optimized, and validated to separate nine antihypertensive drugs by experimental design methodology and the Crossed D-Optimal process. In order to find the most suitable separation conditions, twenty-three experiments were carried out, based on the simultaneous effects of three solvent (methanol, acetonitrile, and water) compositions in combination with different pH values of the mobile phase. The components were cross-combined with a pH factor (quadratic x quadratic process), whereas optimal adjusted models were used for the eight individually chosen responses. The optimal mobile phase consisted of methanol, acetonitrile, and 0.05 M aqueous ammonium acetate at pH 3.1 (18:26: 56 v/v/v, pH adjustment with formic acid). Analysis was carried out on a C₁₈ column (150 × 4.6 mm, 5 µm) at 40 °C using photodiode array detection at 242 nm. The system was found to produce sharp and well resolved peaks for all analytes while the retention times ranged from 2.3 to 31 min. The method was linear (r² > 0.999) and reliable since the accuracy (recovery = 100 ± 2.9) and the precision (relative standard deviation < 2%) met International Conference on Harmonization guidelines. The technique was shown to be a useful tool for separating complex mixtures using experimental design methodology.     

Erratum to: Multiple Response Optimization of a HPLC Method for the Determination of Enantiomeric Purity of S-Ofloxacin

The aim of the study was to develop a new HPLC method for direct chiral separation of Ofloxacin enantiomers using polar non-aqueous mobile phase by application of response surface methodology. Rotatable central composite design (CCD) with eight factorial points, six axial points and six replications in central point was used to evaluate the influence of three independent variables (concentration of methanol, diethylamine and flow rate) on the output responses (capacity factor of first peak, tailing factors of both the enantiomers, resolution between the Ofloxacin enantiomers, retention time of the last peak and chromatographic optimization function). Further, CCD data were combined with multiple response optimization in order to obtain a set of optimal experimental conditions (% methanol/hexane/acetonitrile-43.33/10/46.62 (v/v), % acetic acid/diethylamine-0.4/0.2 and flow rate as 1.4 mL min⁻¹) leading to the most desirable compromise between resolution and analysis time. The method demonstrated good correlation between observed and predicted responses. The developed method was validated according to ICH guidelines and applied for quantitative analysis of two commercially available tablets Zenoflox (Ofloxacin) and Glevo (Levofloxacin). Good agreement was found between the assay results and the label claim of the marketed formulations by showing good %recovery and %CV. The study resulted in a better chromatographic system for the determination of Ofloxacin enantiomers.

     

Development and Validation of a Stability-Indicating LC Method for Determination of Ebastine in Tablet and Syrup

A simple stability-indicating reversed-phase liquid chromatographic method with diode-array detection was developed and validated for the quantitative determination of ebastine in tablets and syrup. The LC method was carried out on a C₁₈ column with acetonitrile:phosphoric acid 0.1% pH 3.0 (55:45, v/v) as mobile phase, at a flow rate of 1.2 mL min⁻¹. Ultraviolet detection of ebastine was at 254 nm. A linear response (r = 0.9999) was observed in the range of 10–80 μg mL⁻¹. The RSD values for intra- and inter-day precision studies showed good results (RSD < 2%) and accuracy was greater than 98%. Validation parameters such as specificity and robustness were also determined. The method was found to be stability-indicating and can be applied to quantitative determination of ebastine in tablets and syrup.

     

Multiple Response Optimization of a HPLC Method for the Determination of Enantiomeric Purity of S-Ofloxacin

The aim of the study was to develop a new HPLC method for direct chiral separation of Ofloxacin enantiomers using polar non-aqueous mobile phase by application of response surface methodology. Rotatable central composite design (CCD) with eight factorial points, six axial points and six replications in central point was used to evaluate the influence of three independent variables (concentration of methanol, diethylamine and flow rate) on the output responses (capacity factor of first peak, tailing factors of both the enantiomers, resolution between the Ofloxacin enantiomers, retention time of the last peak and chromatographic optimization function). Further, CCD data were combined with multiple response optimization in order to obtain a set of optimal experimental conditions (% methanol/hexane/acetonitrile-43.33/10/46.62 (v/v), % acetic acid/diethylamine-0.4/0.2 and flow rate as 1.4 mL min^?1) leading to the most desirable compromise between resolution and analysis time. The method demonstrated good correlation between observed and predicted responses. The developed method was validated according to ICH guidelines and applied for quantitative analysis of two commercially available tablets Zenoflox (Ofloxacin) and Glevo (Levofloxacin). Good agreement was found between the assay results and the label claim of the marketed formulations by showing good %recovery and %CV. The study resulted in a better chromatographic system for the determination of Ofloxacin enantiomers.     

Simultaneous determination of ten anticoagulant rodenticides in tissues by column-switching UHPLC-ESI-MS/MS

This paper describes the development of a method for the simultaneous determination of ten anticoagulant rodenticides (coumafuryl, warfarin, pindone, coumatetralyl, coumachlor, difenacoum, bromadiolone, brodifacoum, chlorophacinone and flocoumafen) in the liver and kidney based on column-switching liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry. The simple sample preparation includes extraction with methanol. A C₁₈ trapping column was used for online solid-phase extraction before analytical separation with the mobile phase comprising a mixture of 0.1 % formic acid in water, methanol and acetonitrile. Chromatographic separation was achieved using a Thermo Hypersil ultra high-performance liquid chromatography (UHPLC) C₁₈ column with the mobile phase consisting of 5 mM ammonium formate buffer (pH?=?9) and methanol. The column-switching procedure ensured no matrix effects during electrospray ionization (ESI). Extraction recoveries ranged between 91 and 100 % for liver and between 89 and 97 % for kidney. The method showed good linearity up to 750 ng g^?1. The limit of detection ranged between 0.001 and 0.022 ng g^?1 for liver and between 0.001 and 0.028 ng g^?1 for kidney. The developed method was successfully used in several animal poisoning cases.     

LC Assay of Eletriptan in Tablets and In Vitro Dissolution Studies

Eletriptan (ELT) is a new selective serotonin agonist approved for the treatment of acute migraine headaches. A simple and rapid liquid chromatographic method was developed and validated for the assay of ELT in tablets. Chromatography was carried out on a 250 mm × 4.6 mm C₁₈ column at 30 °C. Acetonitrile–15 mM triethylamine solution (adjusted to pH 7.0 using concentrated o-phosphoric acid) (60:40, v/v) mixture was used as mobile phase at 1.0 mL min^?1 flow rate and UV detector was set at 225 nm. A linear response (r ²  = 0.9999) was observed in the range of 0.1–1.6 μg mL^?1. The method showed good recoveries (100.08 %) and the RSD values for intra- and inter-day precision were 0.78–1.93 and 1.10–2.15%, respectively. The method can be used for quality control assays and in vitro dissolution studies of ELT in tablets.     

A Validated HPLC Method with Fluorescence Detection for the Determination of Droperidol in Pharmaceutical Tablets,Human Serum,and Human Milk

A sensitive and simple HPLC method with fluorimetric detection has been developed for determination of droperidol in pharmaceutical tablets, human serum, and human milk. Chromatography was performed on a 100 mm × 3 mm i.d. C₁₈ column with methanol–water, 30:70 (v/v), pH 3.5, as mobile phase at a flow-rate of 0.8 mL min⁻¹. The injection volume was 5 μL and detection was by monitoring emission at 324 nm after excitation at 283 nm. Droperidol and p-hydroxybenzoic acid (internal standard) eluted after 5.3 and 6.1 min, respectively. The method was validated over the concentration range 1.14 × 10⁻⁷ to 9.12 × 10⁻⁶ M. Selectivity was good and the limits of detection and quantitation of the method were approximately 3.54 × 10⁻⁸ and 1.07 × 10⁻⁷ M, respectively, corresponding to 13 and 40 ng mL⁻¹. The applicability of the method to determination of droperidol in pharmaceuticals, human serum, and human milk was demonstrated.

     

Liquid chromatography/electrospray ionization/ion mobility spectrometry of chlorophenols with full flow from large bore LC columns

Chlorophenols (CPs) as a mixture of fourteen congeners from mono- to pentachlorophenol were determined using liquid chromatography/electrospray ionization/ion mobility spectrometry (LC/ESI/IMS) to describe the response and analytical performance of a mobility spectrometer as a detector for liquid chromatography. The mobility spectrometer was equipped with an interface so that flows from a large bore column could be electrosprayed directly into the drift tube at flow rates up to 500 μL/min without splitting of flow. A linear gradient of the mobile phase from 40% to 90% methanol and 60% to 10% acetic acid (AcOH)–ammonium acetate buffer solution over 40 min with a C18 column provided baseline separations though mobility spectra for CPs were influenced by mobile phase composition. Product ions formed from CPs with ESI included phenoxide anions CPO^?, AcOH·CPO^?, CPOH·CPO^?, and Na⁺·(CPO^?)₂ and were found to be governed by the drift gas temperature. Ions were identified using LC/ESI/mass spectrometry (MS) and supported by results from computational modeling. Quantitative response was affected by congener structure through the acidities of the OH moiety and by the composition of the mobile phase. Limits of detection ranged from 0.135 mg/L for 2,3,5-trichlorophenol and pentachlorophenol to 2.23 mg/L for 2-chlorophenol; corresponding linear ranges were 20 and 70.