An improved HPLC method with the aid of a chemometric protocol: simultaneous analysis of amlodipine and atorvastatin in pharmaceutical formulations

Statistical experimental design and Derringer's desirability function were applied to develop an improved RP-HPLC method for the simultaneous analysis of amlodipine and atorvastatin in pharmaceutical formulations. Four independent factors were considered: acetonitrile content in the mobile phase; buffer pH; buffer concentration; and flow rate. The preliminary screening step was carried out, according to a 2(4-1) fractional factorial design, to identify the significant factors affecting the analysis time response. Then central composite design was applied for a response surface study, in order to examine in depth the effects of the most important factors. Subsequently, Derringer's desirability function was employed to simultaneously optimize the six responses: retention factor of first peak; two resolutions; and three retention times, each having a different target. This procedure allowed deduction of two separate optimum conditions, intended for the analysis of quality control and plasma samples, within the experimental domain. The predicted optimum for the quality control samples was: methanol-acetonitrile-15 mM K(2)HPO(4) buffer (pH 5.33) (10:42.08:47.92, v/v/v) as the mobile phase and 1.12 mL/min as the flow rate. The method using this optimized condition showed higher sensitivity and shorter analysis time than the previously published reports. The optimized assay condition was validated according to International Conference on Harmonization guidelines.     

Principal component analysis: a chemometric aid for classification of polluted and unpolluted mussels

Ten trace elements (Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd and Pb) were determined in the dissolved ash of the edible part of wild mussels (28 samples) from a polluted site by electrothermal atomic absorption spectrometry. The distribution of the concentration of each element in the mussels was first investigated by means of a test of normality. The correlation matrix around the mean was used as a starting matrix for principal component analysis (PCA). Ten variables were reduced to two principal components, accounting for 77% of the total variance; Al, Cr, Mn, Fe, Zn, Cd and Pb are all positively associated with the first principal component and form a cluster of variables, indicating a common terrigenous origin. Comparison with 43 samples of unpolluted mussels from a nearby hatchery, on the basis of eight common variables (concentration of Mn, Fe, Co, Ni, Cu, Zn, Cd and Pb; 71 samples) by using a two-dimensional plot of PC scores, allows the mussels from the two sites to be differentiated.     

Rapid analysis of multicomponent pesticide mixture by GC-MS with the aid of chemometric resolution

Applications of hyphenated chromatographic techniques, especially GC-MS technique, have been reported in chemical, biological, environmental, agricultural and medical analysis. The complexity of the samples in these fields is still an obstacle for the technique to be practical and the overlapping of the multicomponent signals induces chemometric methods widely employed. In this work, taking the rapid analysis of pesticide mixture as an example, a chemometric approach was proposed for resolution of multicomponent overlapping GC-MS signal. In the method, a mass spectral library of pesticides was organized at first, then target factor analysis (TFA) was employed for testing the existence of a specific pesticide in the multicomponent overlapping GC-MS signal, and finally the chromatographic information of the pesticide was extracted by a non-negative immune algorithm (IA). A GC-MS signal of a 40-component pesticide mixture eluted within 9 min was analyzed by the method. It was found that the mass spectra and chromatographic profiles of almost all the pesticides can be obtained.     

Simultaneous determination of olmesartan medoxomil and irbesartan and hydrochlorothiazide in pharmaceutical formulations and human serum using high performance liquid chromatography

 A simple, selective, sensitive, precise, simultaneous high performance liquid chromatographic analysis of serum samples and commercial tablet formulation containing hydrochlorothiazide, olmesartan medoxomil and irbesartan are reported. Good chromatographic separation was achieved using a μ-Bondapak, C18 column (15 cm×4.6 mm, 5 μm), and a mobile phase consisting of acetonitrile-0.2% acetic acid aqueous solution (50∶50, v/v) at a flow rate of 1.0 mL/min. The ultraviolet detector was set at a wavelength of 260 nm. Hydrochlorothiazide, olmesartan medoxomil, and irbesartan were eluted at 1.2, 3.8, and 4.4 min, respectively. No extraneous materials were found to interfere. The method uses protein precipitation with acetonitrile for the preparation of serum sample. The linear ranges for hydrochlorothiazide, olmesartan medoxomil, and irbesartan were 6.25-18.75, 20-60, and 75-225 ng/mL, respectively. The recoveries of hydrochlorothiazide, olmesartan medoxomil, and irbesartan in spiked samples were all greater than 98%, and their relative standard deviations were less than 2.0%. The limits of detection were 1, 2, and 2 ng/mL for hydrochlorothiazide, olmesartan medoxomil, and irbesartan, respectively, and the limits of quantification were 3 ng/mL, which allow their determination at the expected serum concentration levels.     

Fingerprint analysis of Shankhpushpi for species discrimination by HPLC coupled with chemometric methods

Abstract

Shankhpushpi, traded in the Indian market is having different controversial botanical sources, that is, Convolvulus pluricaulis Choisy (Convulvulaceae), Clitoria ternatea Linn. (Papilionaceae), and Evolvulus alsinoides Linn. (Convulvulaceae). Simple, accurate and precise RP-HPLC-PDA method was established for quality assessment and discrimination of Shankhpushpi samples using chromatographic profiling method. In distinction to the routine method of quality assessment which uses single or dual marker peaks, all chromatographic data (retention times/variables) were used. Fifteen shankhpushpi samples were purchased from the market including authenticated samples of all three C. pluricaulis, C. ternatea, E. alsinoides. A total of 18 samples were analyzed by HPLC and the dataset was then treated with multivariate analyses like PCA and HCA by using MINITAB software. Thus, the developed method was useful in discriminating the Shankhpushpi samples and for the perseverance of quality control.     

Development of a multivariate model with desirability-based optimization for determination of atenolol and hydrochlorothiazide by eco-friendly HPLC method with fluorescence detection

Determination of a widely used antihypertensive combination of atenolol and hydrochlorothiazide was achieved by rapid and eco-friendly high-performance liquid chromatography method combined with fluorescence detection. The response surface methodology is conducive to the complete separation of the two drugs in a shorter analysis time. The separation of the mixture was achieved using an Inertsil C18 analytical column (150 × 4.6 mm, 5 µ). The mobile phase used was ethanol: potassium dihydrogen phosphate at pH 3 (65:35 v/v) and the flow rate was 0.7 mL/min. The fluorescence detector operated at excitation and emission wavelengths of 230 and 310 nm (atenolol) and 270 and 375 nm (hydrochlorothiazide). The linearity of the developed method covered a concentration of atenolol of 0.05–5 µg/mL and a concentration of hydrochlorothiazide of 0.02–1 µg/mL. The greenness of the developed method was evaluated by analytical eco-scale and the recently reported evaluation method, that is, green analytical procedure index, and it was found to be an excellent, sensitive, and green alternative to the reported methods. The developed method was validated according to the ICH guidelines and compared with the reference method. No significant difference was found in terms of accuracy.     

Simultaneous determination of sugars and organic acids in aged vinegars and chemometric data analysis

Aceto Balsamico Tradizionale of Modena (ABTM) is a typical product (PDO denomination) of the province of Modena produced by cooked grape must which undergoes a long ageing period (at least 12 years) in series of wooden casks (batterie). The study of the transformations of this product during ageing is extremely relevant in order to control the authenticity of ABTM towards succedaneous products and mislabelling of age.

This paper presents the results of the investigation of sugars and fixed organic acids in ABTM samples of different ages, coming from different batterie. The analytes were simultaneously determined by a gas chromatographic method optimised for this peculiar matrix.

The method shows good separation and resolution of the investigated chemical species and allows their determination in the concentration ranges reported in brackets: malic (7.6–15.5 g kg⁻¹), tartaric (4.0–9.7 g kg⁻¹), citric (0.6–1.5 g kg⁻¹) and succinic (0.36–0.62 g kg⁻¹) acid and glucose (153–294 g kg⁻¹), fructose (131–279 g kg⁻¹), xylose (011–0.39 g kg⁻¹), ribose (0.078–0.429 g kg⁻¹), rhamnose (0.061–0.195 g kg⁻¹), galactose (0.136–0.388 g kg⁻¹), mannose (0.41–1.46 g kg⁻¹), arabinose (0.33–1.00 g kg⁻¹) and sucrose (0.46–6.84 g kg⁻¹), with mean associated errors ranging from 5 to 19% depending on the analytes.

Moreover, the recovery values are always satisfactory, being close to one for most of the analytes.

Furthermore, in order to assess the degree of variability of the different analytes content with vinegar ageing and the similarity/dissimilarity among series of casks a three-way data analysis method (Tucker3) is proposed. The chemometric technique applied on the data set shows differences between the samples on the bases of their different ageing period, and between the batterie, which traditionally have an own peculiar production procedure.     

Simultaneous kinetic spectrophotometric analysis of five synthetic food colorants with the aid of chemometrics

This paper describes a simple and sensitive kinetic spectrophotometric method for the simultaneous determination of Amaranth, Ponceau 4R, Sunset Yellow, Tartrazine and Brilliant Blue in mixtures with the aid of chemometrics. The method involved two coupled reactions, viz. the reduction of iron(III) by the analytes to iron(II) in sodium acetate/hydrochloric acid solution (pH 1.71) and the chromogenic reaction between iron(II) and hexacyanoferrate(III) ions to yield a Prussian blue peak at 760 nm. The spectral data were recorded over the 500-1000 nm wavelength range every 2 s for 600 s. The kinetic data were collected at 760 nm and 600 s, and linear calibration models were satisfactorily constructed for each of the dyes with detection limits in the range of 0.04-0.50 mg L⁻¹. Multivariate calibration models for kinetic data were established and verified for methods such as the Iterative target transform factor analysis (ITTFA), principal component regression (PCR), partial least squares (PLS), and principal component-radial basis function-artificial neural network (PC-RBF-ANN) with and without wavelet packet transform (WPT) pre-treatment. The PC-RBF-ANN with WPT calibration performed somewhat better than others on the basis of the %RPE_T (∼9) and %Recovery parameters (∼108), although the effect of the WPT pre-treatment was marginal (∼0.5% RPE_T). The proposed method was applied for the simultaneous determination of the five colorants in foodstuff samples, and the results were comparable with those from a reference HPLC method.     

Improved 2-nitrobenzenesulfenyl method: optimization of the protocol and improved enrichment for labeled peptides

We have developed the NBS (2-nitrobenzenesulfenyl) method, a quantitative proteome analysis method utilizing stable isotope labeling followed by mass spectrometry. The potential of this method was reported previously, and the procedure has now been further optimized. Here, we describe a procedure utilizing urea or guanidine hydrochloride as a protein denaturant, in conjunction with an improved chromatographic enrichment method for the NBS-labeled peptides using a phenyl resin column. By using this new protocol, both sample loss throughout the protocol and the elution of unwanted unlabeled peptides can be minimized, improving the efficiency of the analysis significantly.     

Simultaneous determination of trace-levels of alloying zinc and copper by semi-mercury-free potentiometric stripping analysis with chemometric data treatment

Assays of copper and zinc in brass samples were performed by semi-mercury free potentiometric stripping analysis (S-MF PSA) using a thin-film mercury covered glassy-carbon working electrode and dissolved oxygen as oxidizing agent during the stripping step. The stripping peak transients were resolved by chemometrics, which enabled simultaneous determination of both the copper and the zinc concentrations, thereby eliminating the conventional necessary pretreatment of the sample solution, such as initial addition of Ga(III) or solvent extraction of copper. The brass samples were diluted by factors in the range 2 · 10⁴– 5 · 10⁵ which resulted in quantification of the copper and of zinc contents comparable to the specified values within 10%. On the basis of the chemometric treatment, an empirical expression is deduced relating the stripping time to the recorded potential.     

Simultaneous determination of trace-levels of alloying zinc and copper by semi-mercury-free potentiometric stripping analysis with chemometric data treatment

Assays of copper and zinc in brass samples were performed by semi-mercury free potentiometric stripping analysis (S-MF PSA) using a thin-film mercury covered glassy-carbon working electrode and dissolved oxygen as oxidizing agent during the stripping step. The stripping peak transients were resolved by chemometrics, which enabled simultaneous determination of both the copper and the zinc concentrations, thereby eliminating the conventional necessary pretreatment of the sample solution, such as initial addition of Ga(III) or solvent extraction of copper. The brass samples were diluted by factors in the range 2 · 10⁴– 5 · 10⁵ which resulted in quantification of the copper and of zinc contents comparable to the specified values within 10%. On the basis of the chemometric treatment, an empirical expression is deduced relating the stripping time to the recorded potential. Received: 23 December 1997 / Revised: 25 February 1998 / Accepted: 28 February 1998     

Simultaneous determination of silicon and aluminium in ferrosilicon by instrumental neutron activation analysis with the aid of an Ac-Be isotopic neutron source

A method has been developed for the accurate determination of silicon and aluminium by instrumental neutron activation analysis with the aid of an²²⁷Ac-Be isotopic neutron source. The samples are pellets made of 4.6 g of ferrosilicon and 0.9 g of a pelletizing agent. Each pellet is irradiated twice at two different fast-to-thermal neutron flux ratios. Each irradiation is followed by two integral mode countings. This allows correction for manganese activities and calculation of the fraction of the aluminium activity induced in silicon and aluminium. The method has been tested with laboratory-prepared synthetic samples as well as commercially certified ferrosilicon. For ferrosilicon containing 75% Si, a relative precision of 0.7% for the results of the silicon concentration has been obtained. For aluminium the relative precision varied from 2 to 7% for concentrations of 3.5 to 1.5%.     

Determination of olanzapine and desmethylolanzapine in the plasma of schizophrenic patients by means of an improved HPLC method with amperometric detection

Summary An improved HPLC method with electrochemical detection has been developed for the determination of olanzapine and its main metabolite, desmethylolanzapine, in human plasma. Chromatographic separation and analysis were performed on a C₈ reversed-phase column with a mixture of methanol, acetonitrile, and pH 3.7 phosphate buffer as mobile phase; 2-methylolanzapine was used as internal standard. Careful pretreatment of the plasma samples was implemented by means of solid phase extraction (SPE). Response was linearly dependent on concentration and precision was satisfactory over the concentration range 0.5–75.0 ng mL⁻¹ for both analytes. The limit of detection was 0.2 ng mL⁻¹ for both analytes. Application to plasma samples of patients treated with Zyprexa tablets gave good results. Because of its sensitivity and selectivity, and the need for small plasma samples, this method seems to be a useful tool for clinical monitoring.     

Simultaneous determination of aliskiren and hydrochlorothiazide from their pharmaceutical preparations using a validated stability‐indicating MEKC method

A stability‐indicating MEKC method was developed and validated for the simultaneous determination of aliskiren (ALI) and hydrochlorothiazide (HCTZ) in pharmaceutical formulations using ranitidine as an internal standard (IS). Optimal conditions for the separation of ALI, HCTZ and its major impurity chlorothiazide (CTZ), IS and degradation products were investigated. The method employed 47 mM Tris buffer and 47 mM anionic detergent SDS solution at pH 10.2 as the background electrolyte. MEKC method was performed on a fused‐silica capillary (40 cm) at 28°C. Applied voltage was 26 kV (positive polarity) and photodiode array (PDA) detector was set at 217 nm. The method was validated in accordance with the ICH requirements. The method was linear over the concentration range of 5–100 and 60–1200 μg/mL for HCTZ and ALI, respectively (r²>0.9997). The stability‐indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using the PDA detection. Precision and accuracy evaluated by RSD were lower than 2%. The method proved to be robust by a fractional factorial design evaluation. The proposed MEKC method was successfully applied for the quantitative analysis of ALI and HCTZ both individually and in a combined dosage tablet formulation to support the quality control.     

Alkylation of acylmetalates with alkyl halides to prepare Fischer carbene complexes: an improved protocol

Alkoxy Fischer carbene complexes have been synthesized by alkylation of lithium acylmetalates with alkyl halides in the presence of catalytic amount (5-10 mol %) of n-tetrabutylammonium bromide (n-Bu₄NBr) restricting the temperature below 55 °C to minimize decomposition of the product. The reaction occurs in a biphasic condition involving water and alkyl halide. The effect of cesium on this alkylation reaction has been studied. The presence of a radical quencher, di-tert-butyl phenol, neither affects the yield nor leads to the formation of dimer of di-tert-butyl phenol, which rules out the possibility of radical pathway mechanism. The kinetic study and the ¹H NMR spectra of products suggest an S_N2 pathway particularly involving alkyl halides.     

Development and validation of an improved liquid chromatographic method for the analysis of oxytetracycline

Summary An improved LC method is described for the separation of oxytetracycline and its impurities. The separation is much better than that obtained with official pharmacopoeia methods. The method uses XTerra RP-18, 5 μm (25cm×4.6 mm I.D.), a silica-based stationary phase with methyl end-capping, claimed to reduce silanol activity. The column temperature is set at 30°C and a UV detection is performed at 280 nm. Mobile phase containing acetonitrile −0.25 M tetrabutylammonium hydrogen sulfate pH 7.5−0.25 M ethylenediaminetetraacetic acid pH 7.5-water (115:360:160:365,v/v/v/v) is used at a flow rate of 1.0 mL.min⁻¹, to separate the impurities present in oxytetracycline base. A central composite experimental design is used to optimize the separation. A second isocratic method with higher content of acetonitrile is needed to separate the more retained impurities present only in oxytetracycline hydrochloride. The method is robust and shows good selectivity, repeatability, linearity and sensitivity.     

Development and validation of an improved liquid chromatographic method for the analysis of dirithromycin

The official method for the determination of dirithromycin and related substances in the European Pharmacopoeia (Ph. Eur.) and in the United States Pharmacopeia (USP) is an isocratic liquid chromatographic (LC) method using an ODS column. With this method, the separation of the main component dirithromycin from its epimer is not complete. Moreover, this method suffers sometimes from drift of the baseline and from subsequent quantitation problems. The required resolution is not easy to obtain.

Using an adapted method derived from the one prescribed in the pharmacopoeias, the selectivity of a set of more than 40 reversed-phase columns towards dirithromycin components was investigated. The selection of the most suitable column was achieved by the chromatographic response function (CRF) approach. Several changes were introduced to the method in order to improve the separation and to overcome the baseline drift problem. The resulting method uses a Zorbax Extend column maintained at 30 °C and a mobile phase containing acetonitrile, methanol, 2-propanol, water and a phosphate buffer at pH 7.5. The method allows a good separation of dirithromycin components, which is much better than that obtained with the existing methods. Several impurities of unknown identity are also separated. The method shows good repeatability, linearity and sensitivity, and it is robust. In addition, it proved to be applicable to a wide number of C18 reversed-phase columns.     

An improved HPLC system for the analysis of photosynthetic pigments

Summary A high performance liquid chromatography system for the analysis of photosynthetic pigments is presented. The method employs an octadecylsilica stationary phase, a programmed quaternary mobile phase consisting of mixtures of methanol, acetonitrile, water and hexane, and a photodiode array detector. Carotenoids and chlorophylls are rapidly analysed in a single chromatographic separation. Thecis-trans isomers of most carotenoids are separated by this method.     

Tellurium tetrachloride: an improved method of preparation

An efficient and practical synthesis of tellurium tetrachloride from elemental tellurium and sulfuryl chloride is described.