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.     

Simultaneous Spectrophotometric Estimation of Rabeprazole Sodium and Itopride HCl

Abstract

Two simple, rapid, accurate, and economical analytical methods have been developed for the simultaneous estimation of Rabeprazole Sodium and Itopride Hydrochloride in combined capsule dosage form. First method is based on the determination of Q‐value and second method is based on simultaneous equation method. Rabeprazole Sodium has absorbance maxima at 284 nm and Itopride Hydrochloride has absorbance maxima at 258 nm in methanol AR. The absorption ratio (Q‐value) was determined at 266.6 nm (Iso‐bestic point) and 284 nm (λmax of Rabeprazole Sodium). Both the drugs obey Beer's law in the concentration ranges employed for these methods. Both the methods were found to be simple, rapid, accurate, and can be adopted in routine analysis of drugs in formulations. The accuracy and reproducibility of the proposed method was statistically validated by recovery studies     

Simultaneous Determination of Monoamines,Their Precursor Amino Acids,and Related Metabolites in Mice Brain by High‐Performance Liquid Chromatography with a Coulometric Electrode Array System

Abstract

A method based on high performance liquid chromatography with a coulometric electrode array system (HPLC‐coulometric electrode array) using C₁₈ column has been developed for the simultaneous determination of norepinephrine (NE), epinephrine (E), l‐3,4‐dihydroxyphenylalanine (l‐DOPA), p‐tyrosine (p‐TYR), dopamine (DA), m‐tyrosine (m‐TYR), 5‐hydroxytryptamine (5‐HT), homovanillic acid (HVA), and 5‐hydroxyindoleacetic acid (5‐HIAA) in mice brain. The chromatography was performed using a C₁₈ column (250 mm×4.6 mm i.d. and 5 µm) with sodium acetate buffer (pH 5.0, 0.05 M) and methanol as the mobile phase. Elution of analytes was carried out at a flow rate of 1.0 ml/min. The nine compounds were monitored using an ESA electrochemical detector. Potentials of three electrodes in series were set at 200, 500, and 700 mV, respectively. Optimization of the pH of the mobile phase and the proportion of methanol were also considered. The minimal detection limits were 2–8 ng/ml. Linear (r=0.99) detector performances were observed within a range of 10～2000 ng/ml. Recoveries for the nine compounds in spiked samples were over 90% and the relative standard deviations (RSD) were less than 4.0%.     

Determination of Mycophenolic Acid (MPA) and Its Acyl and Phenol Glucuronide Metabolits Simultaneously in Human Plasma by a Simplified HPLC Method

Abstract

A simple HPLC method with ultraviolet detection for simultaneous determination of Mycophenolic acid (MPA), its phenol glucuronide metabolite (MPAG) and acyl‐MPAG (AcMPAG) in human plasma was established. The plasma samples were prepared with protein‐preciptaing reagent, and the supernatant was eluted on Zorbax column (250 mm×4.6 mm i.d, 5 µm) with 20 mmol/l NaH₂PO₄ buffer (pH 3.0, adjusted with 20% phosphoric acid) and methanol (45:55, v/v) at 304 nm. The column temperature was 45°C, and the flow rate was 1.2 ml/min. The assay was linear within the range of 0.2–50 µg/L for MPA (r=0.9997), 2.8–531 µg/L for MPAG (r=0.9999), and 0.3–24 µg/L for AcMPAG (r=0.9994). Mean absolute recovery of MPA and its metabolites and internal standard was >80%. The average recoveries of MPA, MPAG, and AcMPAG were 94.0–101.4, 98.4–101.9, and 96.1–104.2%, respectively. The RSD of within‐day and between‐day were all lower than 15%. The method described is sensitive, reproducible, and will be useful in TDM or pharmacokinetic studies of MPA.     

Simultaneous Separation and Determination of Lamivudine and Zidovudine in Pharmaceutical Formulations Using the HPTLC Method

Abstract

A high-performance thin-layer chromatographic method (HPTLC) for the simultaneous determination of lamivudine and zidovudine in a binary mixture has been developed. The method developed was based on HPTLC separation of the two drugs followed by densitometric measurements of spots at 276 and 271 nm for lamivudine and zidovudine, respectively. Separation was carried out on Merck HPTLC silica-gel 60 F₂₅₄ plates, using toluene/chloroform/methanol (1:6:3 v:v) as the mobile phase. Validation of the method was performed based on The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines in terms of linearity, accuracy, precision, limit of detection, limit of quantification, and robustness. Second-order polynomial equations were obtained for the regression line in the ranges of 250–1400 and 250–1700 ng/spot for lamivudine and zidovudine respectively. Correlation coefficient (r) values were 0.9998 for both analytes. The method provides sufficient accuracy as indicated by recovery percentages given for lamivudine and zidovudine. For system precision study, the low coefficient of variation values (<2%) for both lamivudine and zidovudine ensured reproducible performance of the instrument. In the method precision study, coefficients of variation <2% were obtained, which showed that the proposed method provides acceptable intraday and interday variation. The detection and quantification limits and were 3.06 and 9.28 ng/spot for lamivudine and 3.34 and 10.13 ng/spot for zidovudine, respectively. Parameters such as mobile-phase composition, volume of mobile phase, time from spotting to development, and time from development to scanning were employed while testing for robustness of the method, and the standard deviation of peak areas was calculated for each parameter. The low coefficient of variation values indicated the robustness of the method. Statistical manipulation did not show any significant effect of one parameter over the others on the robustness of the method.     

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.     

High‐Performance Liquid Chromatographic Determination of Parecoxib in Human Plasma and Pharmaceutical Formulations

Abstract

A simple and sensitive RP‐HPLC method for the determination of parecoxib (PXB) in human plasma and pharmaceutical formulations has been developed and validated. The separation of PXB and the internal standard, ibuprofen (IBF) was achieved on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) column using UV detector at 200 nm. The mobile phase consisted of acetonitrile‐water (92:8 v/v). The linear range of detection was found to be 0.9–18.4 µg/ml (r=0.9985). Intra‐ and inter‐day assay relative standard deviations were observed to be less than 0.3%. The method has been applied successfully for the determination of PXB in spiked human plasma and pharmaceutical preparations. Analytical parameters were calculated and complete statistical evaluation is incorporated.     

Determination of Sumatriptan and Zolmitriptan in Presence of Their Corresponding Degradation Products by HPTLC Methods

Accurate, sensitive, and precise high performance thin layer chromatographic (HPTLC) methods were developed and validated for the determination of sumatriptan and zolmitriptan in presence of their degradation products. Sumatriptan was separated from its degradation products and analyzed on TLC silica gel 60 F₂₅₄ plates using chloroform–ethyl acetate–methanol–ammonia (4:3:3:0.1, v/v) as a developing system followed by densitometric measurement of the bands at 228 nm. Zolmitriptan was determined using chloroform–ethyl acetate–methanol–ammonia (3:3:3:1, v/v) as a developing system followed by densitometric measurement at 222 nm. The methods were validated over a range of 0.5–4 μg/spot for sumatriptan and 0.5–3 μg/spot for zolmitriptan. The proposed methods were successfully applied for the determination of the studied drugs in bulk powder and in their pharmaceutical formulations.     

Rapid Determination of Coumatetralyl in Human Serum by High‐Performance Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry

Abstract

A rapid, sensitive, and selective high‐performance liquid chromatography‐tandem mass spectrometric method (HPLC‐MS‐MS) for the determination of coumatetralyl in human serum using warfarin as an internal standard has been developed and validated. Coumatetralyl and the internal standard were extracted from the human serum samples by liquid‐liquid extraction with ethyl acetate, followed by separation on a XDB C₁₈ reversed‐phase column (150 mm×2.1 mm i.d., 5 µm) using a mobile phase consisting of acetic acid‐ammonium acetate (5 mmol/L, pH=4.5)/methanol (20:80, v/v) at a constant flow rate of 0.40 mL/min. Coumatetralyl and the internal standard were ionized by negative ion pneumatically assisted electrospray and detected in the multiple‐reaction monitoring mode using precursor→product ion combinations at m/z 291→247 and 307→161, respectively. The calibration curve was linear (r²=0.9945) in the concentration range of 0.5～100.0 ng/mL, with a lower limit of quantification of 0.5 ng/mL in human serum. Intra‐ and inter‐day relative standard deviations were less than 6.3 and 11.0%, respectively. The mean extraction recovery was 87.9% for coumatetralyl and 90.1% for the internal standard. This method is found to be able to determine trace coumatetralyl in human serum and can be used for the diagnosis of poisoned human beings.     

HPLC‐UV Detection for Analysis of p‐Benzoquinone Dioxime and p‐Nitrosophenol,and Chromatographic Fingerprint Applied in Quality Control of Industrial p‐Benzoquinone Dioxime

Abstract

A reliable reversed‐phase high performance liquid chromatographic (HPLC) method has been developed for simultaneous determination of p‐benzoquinone dioxime (BQD) and its related impurity p‐nitrosophenol (NSP). Separation was achieved on a Kromasil C₁₈ column by using methanol‐water‐NH₄Ac‐NH₃ solution (pH=7.0, 50 mM) (30/50/20, v/v/v) as the mobile phase, and detection was operated by UV absorption at a wavelength of 305 nm. The method was seen to have good linearity, accuracy, and precision for the concentration range and to be an attractive choice for the quality control of BQD for industrial use. Moreover, the HPLC‐UV‐vis fingerprint of BQD has been established, and successfully applied to quality control of industrial BQD in laboratories of some rubber factories in China. Chromatographic fingerprints of intermediates would become an effective strategy for accelerating the progress of fine chemical industry.     

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.     

Simultaneous Determination of Hydrochlorothiazide and Valsartan in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry

Abstract

A rapid and specific liquid chromatography/tandem mass spectrometry method was described for the simultaneous determination of hydrochlorothiazide and valsartan in human plasma. After extracted from plasma using methanol, hydrochlorothiazide, valsartan and hydroflumethiazide, irbesartan, used as the internal standard, respectively, were chromatographically analyzed on a Phenomenex Kromasil C₈ column with water and methanol (27:73, v/v) as the mobile phase. Selected reaction monitoring was specific for mass detection employing negative electrospray ionization. The calibration standards were linear over the concentration range (3.13–800 ng/ml for hydrochlorothiazide and 11.72–3000 ng/ml for valsartan). The method was found to be suitable for application to a pharmacokinetic study after oral administration of dispersible tablet containing 12.5 mg hydrochlorothiazide and 80 mg valsartan to 20 healthy volunteers.     

Reversed-Phase High Performance Liquid Chromatographic and Thin Layer Chromatographic Methods for the Simultaneous Determination of Benazepril Hydrochloride and Hydrochlorothiazide in Cibadrex Tablets

ABSTRACT

Two procedures were developed for simultaneous determination of benazepril hydrochloride (I) and hydrochlorothiazide (II) in pure, laboratory made mixtures and in pharmaceutical dosage form “Cibadrex tablets® using reversed phase high performance liquid chromatographic and thin layer chromatographic methods.

For reversed phase HPLC, a new very sensitive, rapid, selective method was developed. The linearity ranges were 32-448 ng/20 μl and 40-560 ng/20 μl for benazepril hydrochloride and hydrochlorothiazide, respectively. The corresponding recoveries were 99.38 ± 1.526 and 99.2 ± 1.123.

The minimum detection limits were 7 ng/20 μl and 14 ng/20 μl for benazepril hydrochloride and hydrochlorothiazide respectively.

On the other hand, a new, simple, sensitive and fast thin layer chromatographic scanning densitometric method was developed for simultaneous determination of benazepril hydrochloride and hydrochlorothiazide using ethyl acetate: methanol: ammonia (85: 20: 10 v/v) as the developing system. The R_f values were 0.33 & 0.68 for benazepril hydrochloride and hydrochlorothiazide respectively. The minimum detection limit obtained was 0.12 μg/spot for benazepril hydrochloride and 0.24 μg/spot for hydrochlorothiazide. The mean percentage recoveries were 100.04 ± 1.102 and 99.31 ± 1.009 for benazepril hydrochloride and hydrochlorothiazide respectively.

The two proposed methods were simple, precise, sensitive and could be successfully applied for the determination of pure, laboratory made mixtures and pharmaceutical dosage forms. The results obtained were compared with those obtained by A ^1%.     

Development and validation of high-performance thin-layer chromatographic method for determination of amygdalin

ABSTRACT

A new method for the extraction and quantitative determination of amygdalin has been proposed. Accelerated solvent extraction was applied for the extraction, and reversed-phase high-performance thin-layer chromatography method was developed, validated, and applied for the determination of amygdalin in the extracts of apricot, plum, almond, and peach kernels. The chromatographic system used was RP-18 silica, as stationary phase and acetonitrile/water (50:50, v/v), as mobile phase. Densitometric scanning was performed at 210 nm. The method was validated with respect to specificity, linearity, precision, and accuracy. The results showed that the peak area responses were linear within the concentration range of 2.5–50.0 µg/spot (R² = 0.9984). The limit of quantification was 4.28 µg/spot, and the detection limit 1.28 µg/spot. The intra-day and inter-day reproducibility, in terms of %RSD, were in the range of 0.81–1.15 and 1.32–1.89, respectively. The accuracy data were in the range from 99.98 to 100.56%. The method is linear, quantitative and reproducible, and could be used as an efficient and economical green chromatographic procedure for the determination of amygdalin in the fruit kernel.     

Liquid Chromatography‐Electrospray Ionization Mass Spectrometric Method for Determination of Gliclazide in Human Plasma

Abstract

A rapid, sensitive, and specific liquid chromatography‐electrospray ionization mass spectrometric (LC‐ESI‐MS) method has been developed for quantification of gliclazide in human plasma. The analyte and tolbutamide (internal standard, I.S.) were extracted from plasma samples with n‐hexane–dichloromethane (1:1, v/v) and analyzed on a C₁₈ column. The chromatographic separation was achieved within 4.0 min by using methanol–0.5% formic acid (80:20, v/v) as mobile phase and the flow rate was 1.0 mL/min. Ion signals m/z 324.0 and 271.0 for gliclazide and internal standard were measured in the positive mode, respectively. The method was linear within the range of 2.5–2000 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra‐ and inter‐day precisions were lower than 2.8% in terms of relative standard deviation (RSD). The inter‐day relative error (RE) as determined from quality control samples (QCs) ranged from ?1.93% to 1.85%. This validated method was successfully applied for the evaluation of pharmacokinetic profiles of gliclazide modified‐release tablets in 20 healthy volunteers.     

LC–MS Method for the Sensitive Determination of Metoclopramide: Application to Rabbit Plasma, Gel Formulations and Pharmaceuticals

To support real biological sample application, a simple, selective and rapid LC–MS method has been developed and validated for the sensitive determination of metoclopramide in rabbit blood, ex vivo permeation studies and pharmaceutical dosage form. LC–MS analysis was performed isocratically on a Zorbax SB-C₁₈ column with a mobile phase consisting of methanol:ammonium acetate buffer (pH 3.0) 75:25 (v/v) at a flow rate of 0.70 mL min^?1. The outlet of the column was connected to a single quadrupole mass spectrometer with positive mass spectrometric detection. Ions were detected in the positive multiple reaction monitoring mode. The assay was linear over the concentration range of 1.25–200 pg μL^?1 with a limit of detection of 0.077 pg μL^?1 for standard solutions and 2.5–200 pg μL^?1 with a limit of detection of 0.42 pg μL^?1 for serum samples. The method is applicable, covering a variety of pharmaceutical and biological studies. Metoclopramide was extracted from rabbit blood by liquid–liquid extraction using ether as the extraction solvent. The reproducibility of the method was found to be between 0.96 and 1.98 % (RSD) values. The proposed method has been extensively validated for the determination of metoclopramide in all working media. The sample preparations, flow rate and run time of the analytical systems are not time consuming. Moreover, for the stability of metoclopramide, the effect of temperature, UV light, H₂O₂, HCl and NaOH were also investigated.     

Simultaneous Estimation of Ceftazidime and Ceftizoxime in Pharmaceutical Formulations by HPLC Method

The aim of the present study was to develop a fast, sensitive and reliable method for rapid screening of cephalosporin injectable dosage forms namely ceftazidime and ceftizoxime to the detection of counterfeit and substandard drugs that might be illegally commercialized. Ceftazidime, ceftizoxime and cefixime (IS) were separated in a X-Terra RP-18 column (250 × 4.60 mm ID × 5 ??) and DAD detector set at 290 and 260 nm. The mobile phase consisted of a mixture of methanol:water 20:80 (v/v) at a flow rate of 1.0 mL min^?1. Additionally, in order to find the optimum pH value of separation the pK _a values of studied compounds were determined by using two different methodologies. Aqueous pK _a values of studied compounds have been determined by UV-spectrophotometry and liquid chromatography were used for the determination and direct characterization of the dissociation constants by using the dependence of the capacity factor on the pH of the mobile phase in 20% (v/v) methanol?Cwater binary mixture in which separation was performed. The pH of the mobile phase was adjusted with 25 mM H₃PO₄ to 3.2. The method was shown to be linear, sensible, accurate, and reproducible over the range of analysis and it can be used to pharmaceutical formulations containing a single active ingredient within a short analysis time.     

Spectrophotometric Determination of Cadmium(II) Using p,p′‐Dinitro‐SYM‐Diphenylcarbazid in Aqueous Solutions

Abstract

A sensitive and selective spectrophotometric method is proposed for the rapid determination of cadmium(II) using, p,p′‐dinitro‐sym‐diphenylcarbazid, directly in aqueous solution. The reaction between cadmium(II) and p,p′‐dinitro‐sym‐diphenylcarbazid occurs immediately in strong basic media (0.02 N sodium hydroxide solution). The complex shows a maximum of absorption at 630–640 nm, and the absorbance remains stable for at least 24 h. The method allows the cadmium determination over the range 0.5–6.0 µg mL^?1, with a molar absortivity of 2.05×10⁴ L mol^?1 cm^?1 and features a detection limit of 0.13 ppm. The interferences caused by several ions [Ca(II), K(I), Ba(II), Al(III), Pb(II), Zn(II), Cl^?1, NO₃ ^?, SO₄ ^2?], which are present in most of environmental samples, were determined. The validation of the spectrophotometric method was done by recovery test of cadmium(II) in tap water and sea water. The results show that the proposed method has been successfully applied to the determination of cadmium(II) in water samples.     

Spectroscopic Probe–Aluminum(III)–Chrome Azure S Enhanced Determination of Serum Albumin in Microemulsion Medium

Abstract

The sensitizing effect of spectroscopic probe and microemulsion media on the determination of serum albumin by spectrometry was developed. The main factors affecting the determination were investigated in detail. The results showed that serum albumin reacted with spectroscopic probe Al(III)‐CAS to form a blue complex at pH=3.0 in the p‐octyl polyethyleneglycolphenyether microemulsion (Triton X‐100‐M). The λ_max=641 nm, and Beer's law was obeyed in the range of 0.0 and 35.0 µg · ml^?1 for bovine serum albumin (BSA). The sensitizing effect of spectroscopic probe in Triton X‐100-M microemulsion was higher than that in aqueous media. The proposed method has been used to determine protein in serum with satisfactory results.     

Simple,Sensitive, and Rapid LC–MS Method for the Quantitation of Indapamide in Human Plasma—Application to Pharmacokinetic Studies

Abstract

A sensitive and specific liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) method was developed and validated for the identification and quantification of indapamide in human plasma. After the addition of the internal standard (IS) and 0.1 M sodium hydroxide solution, plasma samples were extracted with diethyl ether. The organic layer was evaporated under a stream of nitrogen at 40°C. The residue was reconstituted with 200 µL methanol. The compounds were separated on a stainless‐steel column (C₁₈ Shim‐pack 5 µm 150 mm×2.0 mm I.D., Shimadzu) at a flow rate of 0.2 mL/min by a gradient elution. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring (SIM) mode via an electrospray ionization (ESI) source. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 0.5–100.0 ng/mL with a coefficient of determination (r) of 0.9998 and good back‐calculated accuracy and precision. The intra‐ and inter‐day precision (RSD%) was lower than 10% and accuracy ranged from 85% to 115%. The lower limit of quantification was identifiable and reproducible at 0.2 ng/mL with 0.2 mL plasma. The proposed method enables the unambiguous identification and quantification of indapamide for pharmacokinetic, bioavailability, or bioequivalence studies.