Determination of UV-transparent compounds by liquid chromatography using the UV detector

An indirect UV photometric detection technique is described in which a low concentration of a UV-absorbing compound (UVAC) is added to the mobile phase in reversed phase liquid chromatography, thereby making it possible for non UV-absorbing compounds such as the lower alcohols to be detected by the UV detector. This happens because the injected analyte may extract a portion of the UV absorbing compound from the mobile and/or stationary phase and the complex is co-eluted as a positive peak at the retention time of the analyte. Alternatively, the injected analyte may appear as a negative peak if the UV-absorbing compound is transferred to the mobile and stationary phases. In any case, the injected compound appears either as a positive or negative peak depending on the relative polarities and concentrations of all the compounds in the system. In addition, the resulting excess or deficiency of detection agent in the stationary phase is eluted separately as a positive or negative peak, indicating that the system has returned to equilibrium. In the work described herein, the chromatographic conditions and variables of the indirect photometric technique were studied to develop a quantitative HPLC method for UV-transparent compounds. It was found that under optimal conditions it is possible to determine some analytes quantitatively at concentrations as low as 0.05%.     

Determination of phenolic antioxidants in pharmaceutical formulations by liquid chromatography and migration study on HDPE packagings

Summary The performance of UV diode-array, spectrometric and electrochemical detectors was compared in the chromatographic analysis of trace amounts of six phenolic antioxidants. Quantitative validation was undertaken; the linearity range was wider using UV detection although the limits of detection were lower with electrochemical detection. UV detection was applied both to identification of an antioxidant in a hydrophilic suspension and to a migration study.     

Determination of polyphenols in wines by liquid chromatography with UV spectrophotometric detection

This paper describes a new chromatographic method for the determination of polyphenolic compounds in wines. The method is based on the separation of analytes by reversed-phase mode in a C18 column (2.6 μm particle size) and UV absorption spectroscopy. The elution gradient is generated from 0.1% formic acid aqueous solution and acetonitrile as an organic modifier. Experimental conditions including pH, percentage of organic modifier and elution gradient profile have been thoroughly optimized using experimental design. A multi-objective function has been defined as a criterion for obtaining a satisfactory compromise among number of compounds separated, resolution and analysis time. Multi-detection at 280, 310 and 370 nm has been utilized in order to work under the most appropriate wavelengths for each compound. Figures of merit including linearity ranges, precisions, detection limits and recoveries have been established under selected experimental conditions using synthetic standards and commercial red wines. The method has been applied to analyze red wines from various Spanish regions.     

Determination of nicotinamide by stopped-flow injection method in pharmaceutical formulations

A simple stopped-flow injection system with spectrophotometric detection was proposed for the determination of nicotinamide (NAM) in pharmaceutical formulations. In this system cyanogen chloride formed from the combination of an acidic KSCN with the NaClO streams reacts with injected NAM to form glutaconic aldehyde. Then the product of these three components was coupled with another buffered (pH 3.5) stream of barbituric acid and directed towards the detector. A 45 s after sample injection the pump was stopped for 130 s. During this time the reactants in the flow cell were provided with the required temperature (40 °C) by placing the cell in a home made cell jacket to increase the yield of the polymethine dye product. Eventually, the absorbance of the formed pink color dye was monitored spectrophotometrically at 560 nm and NAM in the concentration range of 1.0–25.0 μg/mL (R = 0.9974 and D.L = 0.5 μg/mL) was determined. The results obtained by this method were compared statistically and agree with those obtained by the method described in the British Pharmacopoeia.     

The advantages of evaporative light scattering detection in pharmaceutical analysis by high performance liquid chromatography and supercritical fluid chromatography

The evaporative light scattering detector (ELSD) has been used in pharmaceutical analysis by liquid and supercritical fluid chromatography. An ELSD equipped with interchangeable interfaces enables the use of various eluents (UV- or non-UV-absorbing) in isocratic or gradient mode. Analyses were performed on several non-UV-absorbing excipients and active substances. The narrow spread of the response factors of the various compounds investigated has indicated that the detector is suitable for direct raw quantitation of unknown samples in stability studies.     

枸橼酸铋雷尼替丁胶囊有效成份的紫外分光光度法的测定

提出了对枸橼酸铋雷尼替丁胶囊有效成份进行紫外分光光度测定的新方法，该方法简便、快速、准确度高，能用于纯度较高的样品分析，可作为枸橼酸铋雷尼替丁胶囊药品质量标准控制方法。     

Determination of pindolol in pharmaceutical formulations by gas chromatography

Summary A gas chromatographic procedure for the quantitative determination of authentic pindolol and pindolol in pharmaceutical formulations (Visken^R) (Sandoz, Basle, Switzerland) has been investigated. Pindolol in tablets was extracted with ethanol before injecting onto the gas chromatograph. The concentrations adopted were in the range of 1 to 3 mg/cm³ pindolol in alcoholic solution and the accuracy obtained was 98.2%±3.7 and 96.4%±3.6 for pure pindolol and its tablet formulation respectively.     

Stereospecific analysis of flurbiprofen and its major metabolites in plasma and urine by chiral-phase liquid chromatography

Summary Direct chiral-phase HPLC methods have been developed for the determination of flurbiprofen and its major metabolites, namely 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen, in biological fluids using a derivatized amylose chiral stationary phase (CSP; Chiral-pak AD). Quantification of all three analytes, both free and conjugated, in urine was carried out following liquid-liquid extraction using tandem ultraviolet (UV) and fluorescence detection. Determination of flurbiprofen and the 4′-hydroxy-metabolite in plasma utilized the same CSP but required modification in the mobile phase composition and sole use of fluorescence detection. The urine assay was linear (r>0.998) between 0.05–10 μg mL⁻¹, 0.1–20 μg mL⁻¹ and 0.01–2 μg mL⁻¹ for the enantiomers of flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. The plasma assay was linear (r>0.997) between 0.1–6 μg mL⁻¹ and 0.01–0.6 μg mL⁻¹ for the enantiomers of flurbiprofen and 4′-hydroxyflurbiprofen respectively. Both assays, typically yielded within- and between-day imprecision and accuracy values less than 10% for the enantiomers of the different analytes. Initial volunteer studies suggest that the disposition of flurbiprofen displays modest enantioselectivity in humans.     

Determination of fosinopril in pharmaceutical formulations containing hydrochlorothiazide by multiwavelength UV spectrophotometry

A sensitive, simple, rapid and precise method for the simultaneous determination of fosinopril (FOS) and hydrochlorothiazide (HCT) in pharmaceutical formulations is presented. These active ingredients are extracted in aqueous solution and measured by multiwavelength UV spectrophotometry using the program QUEST. HCT acts as an internal standard to verify the accuracy of the analysis. Some aspects of the chemical, spectroscopic and thermoanalytical behaviour of FOS are also reported.     

Determination of vitamin A in pharmaceutical preparations by high-performance liquid chromatography with diode-array detection

Summary A very simple high-performance liquid chromatographic method for determination of Vitamin A in pharmaceutical preparations without the need for saponification was developed. A reversed-phase (Nova-Pack C₁₈, 4 m) column was used with a mobile phase of acetonitrile-tetrahydrofuran-water (55378) and a flowrate of 1.5 ml/min. Sample treatment only consisted of the extraction of retinol acetate content from capsules or tablets with methanol. Total extraction was achieved by shaking vigorously with the aid of magnetic stirring for three hours at room temperature. No change of solvent is necessary to introduce the sample in the chromatographic system. This method is suitable for routine quantification of Vitamin A.     

Determination of ambroxol in dog plasma by high-performance liquid chromatography and UV detection

Summary A new sensitive HPLC-UV method has been developed and validated for the determination of amboroxol in dog plasma enabling the investigation of a newly developed 75 mg ambroxol-containing retard capsule of EGIS Pharmaceuticals Ltd., Budapest, Hungary. A gradient method was used for removing the longer retained plasma components of no interest. The separation was performed on a BDS Hypersil C18 (5 μm, 250×2.1 mm) analytical column, supplied with a 10 mm guard column containing the same packing material. The detection was performed at 210 nm. The calibration curve was linear in the range 25–2000 ng·mL⁻¹. Nerisopam (EGIS-6775) was used as internal standard. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Ion chromatography coupled with fluorescence/UV detector: A comprehensive review of its applications in pesticides and pharmaceutical drug analysis

Ion chromatography (IC) is a well-known technique for trace determination of inorganic ions and small organic acids. Recently, it has also appeared as a promising alternative to reverse phase chromatography for the determination of polar pesticides and pharmaceutical drugs in various sample matrices. Therefore, this study aims to provide a comprehensive overview of the application of IC coupled to fluorescence (FLD) or UV detector for the determination of pesticides and pharmaceutical drugs in samples from all walks of life. Apart from advantages and limitations, a short comparison of IC-FLD/UV with other techniques especially reverse-phase chromatography was drawn to envision future research efforts in this direction. Finally, several related areas such as IC hyphenation with different detectors (spectroscopic and spectrometer), miniaturization, automation, green chemistry, mobile phase, column, sample preparation, etc., were discussed to highlight its application for the determination of a wide range of analytes in the complex sample matrix.     

Determination of chloride in biological fluids as pentafluorobenzylchloride by reversed-phase high-performance liquid chromatography and UV detection

Summary A reversed-phase high-performance liquid chromatographic method for the determination of chloride in plasma, urine, saliva, sweat and aqueous solution is described. Chloride, in solution in aqueous acetone, is converted by means of pentafluorobenzyl bromide into pentafluorobenzyl chloride. This derivative is separated on a ODS-5 m reversed-phase column using isocratic elution with acctonitrile/water, 50/50, v/v, at a flow rate of 2.0 ml/min, and detected by a UV detector at 264 nm. The method is rapid, accurate and sufficiently sensitive for the determination of chloride in less than 10 l sample volume of a biological fluid.     

Determination of nitrate in environmental water samples by conversion into nitrophenols and solid phase extraction−spectrophotometry, liquid chromatography or gas chromatography-mass spectrometry

Conversion of nitrate into a nitro-phenol derivative by reaction with 2-methylphenol or 2,6-dimethylphenol allowed at least 100-fold enrichment of the derivative on Lichrolut EN polymeric cartridge, and it is stable for up to 1 month on the cartridge. The derivative could be eluted with ammonia-methanol mixture. This reaction for nitrate determination has permitted a choice of final measurement by UV-Vis spectrophotometry, liquid chromatography or gas chromatography-mass spectrometry when the limits of detection were 10, 6 and 3 μg l⁻¹, respectively, and the calibration range 20 μg to 10 mg l⁻¹ nitrate. The method has been validated by spiking natural water samples, when the recovery of nitrate was 98.5-108.4% (relative standard deviation 2.5-6.1%).     

Ultra-high capacity liquid chromatography chip/quadrupole time-of-flight mass spectrometry for pharmaceutical analysis

Nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) has attracted increasing interest in virtue of high sensitivity, low sample consumption, and minimal matrix effect. In this work a HPLC-Chip/quadrupole time-of-flight (Q-TOF) MS device with a new ultra-high capacity small molecule chip (UHC-Chip) which features a 500 nL enrichment column and a 150 mm × 75 μm analytical column, was evaluated with a drug mixture covering a wide range of polarities. Excellent chromatographic precision with 0.1-0.5% RSD for retention time and 1.7-9.0% RSD for peak area, low limit of detection, good chip-to-chip reproducibility and linearity were obtained by using this UHC-Chip. Compared with the standard HPLC-Chip with 40 nL trapping column, the UHC-Chip showed higher enrichment capability and hence gave a higher response in signal detection. Additionally, 4-30 times increase in sensitivity was obtained compared with conventional LC/MS, which indicated that UHC-Chip/MS was a valuable tool for the quantitative analysis of low level impurities and degradation products in pharmaceuticals. Moreover, satisfactory results obtained from trace drug analysis of serum samples further proved its practicality and potential for use in drug testing and development.     

Determination of piribedil in pharmaceutical formulations by micellar electrokinetic capillary chromatography

A fast and simple micellar electrokinetic capillary chromatographic method was developed for the analysis of piribedil in pharmaceutical formulations. The effects of buffer concentration, buffer pH, sodium dodecyl sulphate (SDS) concentration, organic modifier, applied voltage and injection time were investigated. Optimum results were obtained with a 50 mM borate buffer at pH 8.0 containing 50 mM SDS by using a fused silica capillary (50 m internal diameter, 72 cm effective length). The sample was injected hydrodynamically for 4 s at 50 mbar pressure and the applied voltage was +30 kV. The detection wavelength was set at 205 nm. Diflunisal was used as an internal standard. The analysis was performed at 25 °C and the total run time was 14 min. The method was suitably validated with respect to linearity range, limit of detection and quantification, precision, accuracy, specificity and robustness. The linear calibration range was 5–100 g mL^–1 and the limit of detection was determined as 1 g mL^–1. The method developed was successfully applied to the determination of piribedil in pharmaceutical formulations. The results were compared with a spectrophotometric method reported in the literature and no significant difference was found statistically.     

Determination of risedronate in rat plasma samples by ion-pair high-performance liquid chromatography with UV detector

In the present work, an analytical method for determination of risedronate, a member of bisphosphonates, is described for the routine analysis in rat plasma. Sample pre-treatment involves protein precipitation, co-precipitation with calcium at alkaline pH, hydrolysis of possible derivatives of pyrophosphate and reprecipitation. A good separation was obtained by using a reversed-phase column (Hypersil ODS-2 C₁₈, 4.6 mm × 250 mm, 5 μm). The mobile phase was an aqueous solution of buffer (contained 1.5 mM EDTA-2Na, 1 mM sodium etidronate, 11 mM sodium phosphate and 5 mM tetrabutylammonium bromide as ion-pair reagent) - methanol (88:12, v/v) adjusted to pH 6.75 using 1 M NaOH. The flow rate was 1 ml min⁻¹. UV detection (λ = 262 nm) was used to quantitate risedronate in the concentration range of 10-500 ng ml⁻¹. The limit of detection and quantitation for risedronate were 7 and 10 ng ml⁻¹, respectively. The method was applied successfully to plasma samples from Wistar rats undergoing oral administration of risedronate mini-pills. Precision, extraction recoveries, as well as accuracy results, were satisfactory and no interference was found at the retention time of risedronate. Hence, the method is suitable for monitoring risedronate in rat plasma.     

Determination of water-soluble vitamins in pharmaceutical preparations by reversed-phase high-performance liquid chromatography with a mobile phase containing sodium dodecylsulphate andn-propanol

Summary A reversed-phase liquid chromatographic method with sodium dodecylsulphate-n-propanolwater as mobile phase has been used to separate and determine six water-soluble vitamins in twelve minutes. The analytical characteristics linear range, sensitivity, detection limits, and precision were evaluated. The lowest detection limits were those of nicotinic acid (not usually present in pharmaceutical products), 0.7 mgL⁻¹, nicotinamide, 1.3 mg L⁻¹, and pyridoxine, 1.4 mg L⁻¹. When the method was applied to the determination of the vitamins in pharmaceutical samples the values found agreed with those on the labels.     

Determination of aprotinin by high-performance liquid chromatography

Summary A highly accurate and reproducible method for the determination of aprotinin (bovine pancreatic trypsin inhibitor) by HPLC is described. In the experiments, the relative standard deviation was 1.2% and detection limit 1 FIP-U cm^–3. Also, the method is quick and selective and active ingredients from difference source correlate well with enzymatic method. Analyses at different laboratories can be compared directly.     

Determination of polysorbate 80 in parenteral formulations by high-performance liquid chromatography and evaporative light scattering detection

Drugs that are not very soluble in aqueous formulations are solubilized with surfactants such as polysorbate 80. In order to evaluate the stability of excipient such as polysorbate 80 in drug formulation, a rapid chromatographic methodology is desired; however, polysorbate 80 does not have a strong chromophore for monitoring by absorption spectrometry. A simple and fast method for the analysis of polysorbate 80 in pharmaceutical formulations was developed using high-performance liquid chromatography with evaporative light scattering detection (ELSD). Separation of polysorbate 80 as a single peak was achieved on a C18 column using a methanol/water gradient mobile phase and ELS detection. The method is specific for polysorbate 80 in the formulation as there were no interferences from the drug or other excipients. Precision, recovery, linearity and limit of quantitation/detection experiments gave acceptable results during the evaluation of the method.