Simultaneous determination of diethylene glycol and propylene glycol in pharmaceutical products by HPLC after precolumn derivatization with p-toluenesulfonyl isocyanate

A simple and reliable HPLC method was developed for the simultaneous quantitative analysis of diethylene glycol (DEG) and propylene glycol (PG) in pharmaceutical products by precolumn derivatization. The derivatization reagent p-toluenesulfonyl isocyanate (TSIC, 10 microL, 20% in ACN v/v) was added to 100 microL of the sample, and then 10 muL of water was added. The resulting derivatives were separated using a C(18)analytical column and a mobile phase composed of 0.01 M KH(2)PO(4)buffer (adjusted to pH 2.5 with phosphoric acid) and ACN (47:53 v/v) at 1 mL/min and 25 degrees C. For detection, UV light at 227 nm was used. The derivatization conditions including reaction time, temperature, and concentration of TSIC were optimized. The calibration curves were linear from 0.062 to 18.6 microg/mL (r(2)= 0.9999) and from 0.071 to 21.3 microg/mL (r(2) = 0.9999) for DEG and PG, respectively. The RSD values of intra- and interday assays were all below 4% for DEG and PG. The proposed method was then successfully applied to analyze two Armillarisin A injection samples and two spiked syrup samples.     

Improved method for the determination of zinc pyrithione in environmental water samples incorporating on-line extraction and preconcentration coupled with liquid chromatography atmospheric pressure chemical ionisation mass spectrometry

A method has been developed for the determination of zinc pyrithione (ZnPT) in environmental water samples using monolithic reversed-phase silica columns for rapid on-line large volume solid phase extraction in tandem with on-line matrix removal using sacrificial strong anion exchange (SAX) columns. This is coupled with reversed-phase liquid chromatography with atmospheric pressure chemical ionisation mass spectrometric detection. Limits of detection in spiked river water samples, using a 200 mL preconcentration volume, were determined as 18 ng L(-1), with a limit of quantitation of 62 ng L(-1). The percentage recovery from spiked river water was found to be 72+/-9 (n=3 extractions), whilst overall method precision, following 10 repeat complete analyses was found to be 27% RSD at 1 microg L(-1). Linearity was determined over the concentration range of 0.25-10 microg L(-1) and the calculated regression coefficient was R(2)=0.9802. The method was used to investigate the environmental fate of zinc pyrithione in waters and its partition coefficient between sediment and water phases.     

Development and validation of a new HPLC-UV method for the simultaneous determination of triclabendazole and ivermectin B1a in a pharmaceutical formulation

A rapid, simple, and sensitive RP-HPLC analytical method was developed for the simultaneous determination of triclabendazole and ivermectin in combination using a C18 RP column. The mobile phase was acetonitrile-methanol-water-acetic acid (56 + 36 + 7.5 + 0.5, v/v/v/v) at a pH of 4.35 and flow rate of 1.0 mL/min. A 245 nm UV detection wavelength was used. Complete validation, including linearity, accuracy, recovery, LOD, LOQ, precision, robustness, stability, and peak purity, was performed. The calibration curve was linear over the range 50.09-150.26 microg/mL for triclabendazole with r = 0.9999 and 27.01-81.02 microg/mL for ivermectin with r = 0.9999. Calculated LOD and LOQ for triclabendazole were 0.03 and 0.08 microg/mL, respectively, and for ivermectin 0.07 and 0.20 microg/mL, respectively. The intraday precision obtained was 98.71% with RSD of 0.87% for triclabendazole and 100.79% with RSD 0.73% for ivermectin. The interday precision obtained was 99.51% with RSD of 0.35% for triclabendazole and 100.55% with RSD of 0.59% for ivermectin. Robustness was also studied, and there was no significant variation of the system suitability of the analytical method with small changes in experimental parameters.     

On-line automatic SPE-CE coupling for the determination of biological markers in urine

Automatic SPE has been coupled on-line to CE by a transfer tube and the replenishment system of the CE instrument. The approach allows the target analytes (viz. creatinine, creatine, xanthine, hypoxanthine, uric acid, p-aminohippuric acid and ascorbic acid in urine samples) to be removed from the sample matrix, cleaned up, preconcentrated and injected into the capillary. The detection limits range between 0.14 and 4.50 microg/mL, the quantification limits between 0.45 and 15.0 microg/mL, and linear dynamic ranges - which include the reference healthy human values - from the quantification limits to 1332 microg/mL. The precision, expressed as RSD, ranges between 0.38 and 2.22% for repeatability and between 1.79 and 7.61% for within-laboratory reproducibility. The errors, expressed as RSD for all compounds, range between 0.20 and 6.90%. The time for automatic SPE and that necessary for the individual separation-detection of the target analytes are 13 and 12 min, respectively; the analysis frequency is 5 h(-1). The accuracy of the method and potential matrix effects were studied by using spiked samples and recoveries between 96.00 and 103.07 % were obtained. The proposed method was applied to samples from healthy young students.     

Fast assay of glucosamine in pharmaceuticals and nutraceuticals by capillary zone electrophoresis with contactless conductivity detection

A novel capillary electrophoresis (CE) method with contactless conductivity detection suitable for the determination of glucosamine (GlAm) and K(+) in pharmaceuticals was devised. Under the optimum conditions (aqueous 30 mM acetate buffer of pH 5.2 as the background electrolyte; voltage 30 kV; 25 degrees C), GlAm (migrating as glucosaminium cation) was well separated from K(+) that could occur in the dosage forms as excipient. The CE analysis was performed in fused-silica capillaries (50 microm i.d., 75 cm total length, 27 cm to detector) and the separation took <3 min. The calibration graphs were linear for both GlAm (100-300 microg/mL; r(2)=0.997) and K(+) (15-75 microg/mL; r(2)=0.997) when using ethanolamine (100 microg/mL) as the internal standard. The LOD values (S/N=3) were 9.3 microg/mL for GlAm and 2.9 microg/mL for K(+). The method was applied to the assay of GlAm content in various dosage forms. Intermediate precision evaluated by determining the content of GlAm in a single formulation on 3 consecutive days was characterized by RSD 2.35% (n=15). Acceptable accuracy of the CE method was confirmed by the added/found GlAm recovery experiments (recoveries 94.6-103.3%) and by statistical comparison of the results attained by the proposed CE and a reference HPLC method.     

UV spectrophotometric determination of bioflavonoids from a semisolid pharmaceutical dosage form containing Trichilia catigua Adr. Juss and Ptychopetalum olacoides Bentham standardized extract: analytical method validation and statistical procedures

A precise, accurate, and sensitive UV spectrophotometric method was developed and validated for routine quantification of total bioflavonoids, expressed as rutin, from a topical oil-in-water pharmaceutical emulsion containing the extract of Trichilia catigua Adr. Juss and Ptychopetalum olacoides Bentham. The method was validated experimentally, and the data were treated rigorously by statistical analysis. The following analytical parameters were assessed: linearity, specificity, intra- and interrun precision measured as relative standard deviation (RSD, %), intra- and interrun accuracy (E, %), recovery (Rec., %), limit of detection (LOD, microg/mL), and limit of quantification (LOQ, microg/mL). The UV spectrophotometric method was linear (r = 0.9995) for standard rutin over the concentration range of 5.0-15.0 microg/mL with specificity for total bioflavonoids (expressed as rutin) at 361.0 nm with an absence of interferents from the complex matrix; RSD of < or = 1.79%, intrarun (E = 97.88 +/- 1.75 to 99.0 +/- 0.33%) and interrun (E = 98.38 +/- 1.12 to 100.79 +/- 1.30%) accuracy; Rec. = 98.64 +/- 0.42 to 100.74 +/- 0.41%; LOD = 0.20 microg/mL; and LOQ = 0.30 microg/mL.     

Aflatoxin M1 determination in cheese by liquid chromatography-tandem mass spectrometry

A new method for determining aflatoxin M1 (AFM1) in cheese by liquid chromatography-tandem mass spectrometry has been developed. Two methodologies were compared for sample extraction. The first one involves sample extraction with dichloromethane for hard, aged cheese or acetone for fresh cheese and includes a preliminary matrix solid-phase dispersion-extraction step before solid-phase extraction (SPE) clean-up by a Carbograph-4 cartridge. The second method uses a water/methanol solution (90:10, v/v) extraction at 150 degrees C before clean-up. The average recoveries of AFM1 from samples spiked at levels of 0.25-0.45 microg/kg, were 81-92% and the precision (RSD) ranged from 3 to 7% with the first method, whilst the average recoveries were 79-84%, and RSD ranged from 7 to 15% for the second method. Due to different matrix effect, the quantification limits were 0.019-0.025 microg/kg in the first case and 0.048-0.143 microg/kg in the second one, depending on cheese typology.     

Method validation for the analysis of 169 pesticides in soya grain, without clean up, by liquid chromatography-tandem mass spectrometry using positive and negative electrospray ionization

Part of a comprehensive study on the comparison of different extraction methods, GC-MS(/MS) and LC-MS/MS detection methods and modes, for the analysis of soya samples is described in this paper. The validation of an acetone-based extraction method for analysis of 169 pesticides in soya, using LC-MS/MS positive and negative electrospray ionisation (ESI) mode, is reported. Samples (5 g) were soaked with 10 g water and subsequently extracted with 100 mL of a mixture of acetone, dichloromethane and light petroleum (1:1:1), in the presence of 15 g anhydrous sodium sulphate. After centrifugation, aliquots of the extract were evaporated and reconstituted in 1.0 mL of methanol, before direct injection of the final extract (corresponding with 0.05 g soya mL(-1)) into the LC-MS/MS system. Linearity, r(2) of calibration curves, instrument limit of detection/quantitation (LOD/LOQ) and matrix effect were evaluated, based on seven concentrations measured in 6-fold. Good linearity (at least r(2)> or =0.99) of the calibration curves was obtained over the range from 0.1 or 0.25 to 10.0 ng mL(-1), corresponding with pesticide concentrations in soya bean extract of 2 or 5-200 microg kg(-1). Instrument LOD values generally were 0.1 or 0.25 ng mL(-1). Matrix effects were negligible for approximately 90% of the pesticides. The accuracy, precision and method LOQ were determined via recovery experiments, spiking soya at 10, 50, 100 microg kg(-1), six replicates per level. In both ESI modes, method LOQ values were mostly 10 or 50 microg kg(-1) and more than 70% of pesticides analysed by each mode met the acceptability criteria of recovery (70-120%) and RSD (< or =20%), at one or more of the three levels studied. A fast, easy and efficient method with acceptable performance was achieved for a difficult matrix as soya, without cleanup.     

Rapid and sensitive determination of acrylamide in drinking water by planar chromatography and fluorescence detection after derivatization with dansulfinic acid

On the basis of a novel derivatization, a new planar chromatographic method has been developed for the determination of acrylamide (AA) in drinking water at the ultra-trace level. After SPE, the water extracts were oversprayed on a high-performance thin-layer chromatography (HPTLC) silica gel plate with the derivatization agent dansulfinic acid and derivatized in situ. Chromatography was performed with ethyl acetate and the fluorescent product was quantified at 366/>400 nm. Verification was based on HPTLC-ESI/MS, HPTLC-direct analysis in real-time (DART)-TOF/MS and NMR. The routine HPTLC-fluorescence detection (FLD) method was validated for spiked drinking water. The regression analysis was linear (r >0.9918) in the range of 0.1-0.4 microg/L. LOD was calculated to be 0.025 microg/L and experimentally proved for spiked samples at levels down to 0.05 microg/L (S/N = 6) which was suited for monitoring the EU limit value of 0.1 microg/L in drinking water (0.5 microg/L demanded by World Health Organization (WHO)/US Environmental Protection Agency (EPA)). Within-run precision and the mean between-run precision (RSD, n = 3, three concentration levels each) were evaluated to be 4.8 and 11.0%, respectively. The mean recovery (0.1, 0.2, and 0.3 microg/L) was 96% corrected by the internal standard. The method, in comparison with HPLC-MS/MS showed comparable results and demonstrated the accuracy of the method.     

Determination of residual isobutylene oxide--a genotoxic starting material in a drug substance by static headspace gas chromatography

A sensitive static headspace gas chromatography (sHS-GC) method is developed and validated for the determination of residual isobutylene oxide (IBO)-a genotoxic starting material in a drug substance for Phase I clinical trial studies. The experimental parameters, such as headspace vial pressure, headspace oven temperature, vial equilibration time, column flow rate, and GC oven temperature programs are optimized. Under the optimal conditions, the recovery is between 89.3% and 102.4% for spiked samples at three levels of IBO concentration in triplicates in sample preparations. Limits of quantitation (LOQ) and detection (LOD) of the standard solutions are 0.048 and 0.018 microg/mL, respectively. Linear range from 0.018 to 6 microg/mL is obtained with a correlation coefficient of 0.9999. The method is applied to determine residual IBO in drug substance samples from different batches.     

Immunoaffinity column cleanup with liquid chromatography using postcolumn bromination for the determination of aflatoxins in black and white sesame seed: single-laboratory validation

A single-laboratory validation was conducted to establish the effectiveness of an immunoaffinity column cleanup procedure followed by LC with fluorescence detection for the determination of aflatoxins B1, B2, G1, and G2 in sesame seeds. The sample is homogenized with 50% water (w/w) to form a slurry, then the test portion is extracted with methanol-water (60 + 40, v/v) using a high-speed blender. The sample extract is filtered, diluted with 15% Tween 20 in phosphate-buffered saline solution, and applied to an immunoaffinity column. Aflatoxins are removed with neat methanol, then directly determined by RP-LC with fluorescence detection using postcolumn bromination (Kobra cell). Test portions of blank white sesame seed slurry were spiked with a mixture of aflatoxins to give total levels of 4 and 10 microg/kg. Recoveries for individual and total aflatoxins ranged from 92.7 to 110.3% for spiked samples. Based on results for spiked sesame paste (triplicates at two levels), the RSD for repeatability (RSD(r)) averaged 1.1% for total aflatoxins and 1.4% for aflatoxin B1. The method was demonstrated to be applicable to naturally contaminated samples of black and white sesame seeds obtained from local markets in China.     

High-performance liquid chromatographic method for determination of reversible isomers of SU5416

SU5416 shows light-induced reversible geometric isomerism. A simple, reliable, isocratic HPLC method using an UV-vis detector at lambda(425nm) was developed. The method provides efficient (R(S)=3.5) analysis of the two isomers with retention of the isomeric integrity. Additionally, the method has linearity over a wide range (50-1000microg/mL, r(2)=0.99), is accurate (99-102%, RSD <4%), and reproducible (RSD <0.8%). The method was used for analyzing pharmaceutical samples and understanding the kinetics of SU5416 isomers in methanol. In addition, this method can be used for quantifying the non-isolatable E-isomer.     

Liquid chromatographic analysis of vitamin B6 in reconstituted infant formula: collaborative study

A liquid chromatographic (LC) method was validated for the determination of total vitamin B6 in infant formula. Total vitamin B6 was quantified by converting the phosphorylated and free vitamers into pyridoxine. Pyridoxine was determined by ion pair reversed-phase LC with fluorescence detection. The method was subjected to an AOAC collaborative study involving a factory-manufactured, milk- and soy-based infant formula. Each was spiked at 3 concentrations in the range of 0-1 microg/g and sent as blind duplicate to participant laboratories. Nine laboratories returned valid data which were statistically analyzed for outliers and precision parameters. The repeatability relative standard deviation (RSD(r)) ranges were 2.0-4.0 and 3.5-5.9% for fortified milk- and soy-based formulas, respectively. The reproducibility relative standard deviation (RSD(R)) ranges were 8.2-8.4 and 6.7-11.2% for fortified milk- and soy-based formulas, respectively. HORRAT values ranged from 0.42 to 0.53, indicating that the precision of the method is acceptable. The mean RSD(r):RSD(R) values were 0.60 and 0.55 for milk- and soy-based formulas, respectively. As expected, RSDs for the unfortified samples were higher, but their HORRAT values (0.81 and 2.06) helped define a realistic limit of quantitation as 0.05 microg/g. Recovery data were quantitative and varied between 81.4 and 98.0% (mean = 89.8%) for each of 6 spiked materials.     

Phenolic compounds in field horsetail (Equisetum arvense L.) as natural antioxidants

In this paper, the study of antioxidant activity and phenolic composition of three different extracts (EtOAc, n-BuOH and H(2)O) of field horsetail (Equisetum arvense L.) is presented. The antioxidant activity has been evaluated measuring the total reducing power (expressed by Ascorbate Equivalent Antioxidant Capacity - AEAC), inhibition of lipid peroxidation, and free radical scavenging capacity (RSC) towards 2,2-diphenyl-1-picrylhydrazyl (DPPH radical) and nitric oxide (NO), respectively. In addition, the total flavonoid content (TFC) and phenolic constituents of each extract have been determined. The results obtained show that the highest RSC regarding both DPPH and NO radicals is expressed by EtOAc extract (EC(50)=2.37 microg/mL and EC(50)=90.07 microg/mL, respectively), and the lowest by H(2)O extract (EC(50)=37.2 microg/mL and EC(50)>333.33 microg/mL, respectively). n-BuOH extract showed the highest total reducing power (AEAC=13.40 microg/mL). Differences in the phenolic composition of examined extracts are found comparing the HPLC chemical profiles. Although, isoquercitrin is the main flavonoid in both EtOAc and n-BuOH extracts, a considerable amount of di-E-caffeoyl-meso-tartaric acid was presented in the n-BuOH extract. In H(2)O extract high content of phenolic acids and low percentage of flavonoids were detected.     

Validation assay for total flavonoids, as rutin equivalents, from Trichilia catigua Adr. Juss (Meliaceae) and Ptychopetalum olacoides Bentham (Olacaceae) commercial extract

An ultraviolet spectrophotometric method was validated for total flavonoid quantitation, as rutin equivalents, present in the Trichilia catigua Adr. Juss (Meliaceae) and Ptychopetalum olacoides Bentham (Olacaceae) commercial extract. Parameters as linearity, interval (range), specificity, estimated limit of detection (LOD, microg/mL), estimated limit of quantitation (LOQ, microg/mL), recovery (R, %), precision or relative standard deviation (RSD, %), and accuracy (E, %) were established. The analytical method was validated according to the experimental results: correlation coefficient (r = 0.9997); interval (RSD = 0.15-0.47%; E = 98.98-101.24%); specificity to total flavonoids quantitation, as rutin equivalents, at wavelength 361.0 nm; LOD = 0.09 microg/mL and LOQ = 0.27 microg/mL; R = 99.36-102.14%; adequate intra- and interrun precision (0.30-0.49% and 0.31-0.81%), and intra- and interrun accuracy (100.60-102.38% and 98.58-100.38%).     

Coupling of ionic liquid-based headspace single-drop microextraction with GC for sensitive detection of phenols

A headspace single-drop microextraction (SDME) based on ionic liquid (IL) has been developed for the gas chromatographic determination of phenols. The volume of IL microdrop used was 1 microL. After extraction, the analytes were desorbed from the drop in the injection port and the involatile IL was withdrawn into the microsyringe. To facilitate the withdrawal of IL the upper diameter of the split inlet liner was enlarged to some extent. Some parameters were optimized for the determination of phenols. Under the selected conditions, i.e., desorption for 100 s at 210 degrees C after extraction for 25 min at 50 degrees C in solutions (pH 3) containing 0.36 g/mL sodium chloride, the LODs, RSDs, and the average enrichment factors of phenols were 0.1-0.4 ng/mL, 3.6-9.5% (n=5), and 35-794, respectively. The proposed procedure was applied to the determination of phenols in lake water and wastewater samples, and the spiked recoveries were in the range of 81-111% at a spiked level of 0.4 microg/mL. This method is a promising alternative for the sensitive determination of phenolic compounds.     

Determination of Danshensu, a major active compound of Radix Salvia miltiorrhiza in dog plasma by HPLC with fluorescence detection.

A simple and sensitive high performance liquid chromatography method has been developed for the determination of Danshensu (3, 4-dihydroxyphenyllactic acid) in dog plasma. Plasma samples were extracted with ethyl acetate. Analysis of the extracts was performed on a reversed-phase column with an aqueous phosphate buffer-acetonitrile (93:7, v/v) mobile phase, and 4-hydroxybenzoic acid was used as the internal standard. Fluorescence detection at 285 nm (excitation) and 320 nm (emission) was employed. Standard curves were linear in the range from 0.125 to 11.3 microg/mL (regression coefficient r > 0.993) on three different days. Mean recovery was determined as 96.4% by analysis of plasma standard containing 0.63, 5.65 and 11.3 microg/mL of Danshensu. The inter-day precision (RSD) ranged from 3.4 to 8.6% at concentrations of 0.125, 1.88, 6.28 and 11.3 microg/mL, and the intra-day precision was better than 7.2%. The detection and quantitation limits were 0.063 and 0.125 microg/mL, respectively. This validated assay was applied to the determination of Danshensu concentration in dog plasma after oral administration of Radix Salvia miltiorrhiza extracts.     

Determination of sixteen polycyclic aromatic hydrocarbons in aqueous and solid samples from an Italian wastewater treatment plant

A robust procedure for the determination of 16 US EPA PAHs in both aqueous (e.g. wastewaters, industrial discharges, treated effluents) and solid samples (e.g. suspended solids and sludge) from a wastewater treatment plant (WWTP) is presented. Recovery experiments using different percentages of organic modifier, sorbents and eluting solvent mixtures were carried out in Milli-Q water (1000 mL) spiked with a mixture of the PAH analytes (100 ng/L of each analyte). The solid phase extraction (SPE) procedures applied to spiked waste water samples (1000 mL; 100 ng/L spiking level) permitted simultaneous recovery of all the 16PAHs with yields >70% (6-13% RSD). SPE clean up procedures applied to sewage and stabilized sludge extracts, showed percent recoveries in the range 73-92% (7-13% RSD) and 71-89% (7-12% RSD), respectively. The methods were used for the determination of PAHs in aqueous and solid samples from the WWTP of Fusina (Venice, Italy). Mean concentrations, as the sum of the 16PAHs in aqueous and suspended solid samples, were found to be approx. in the 1.12-4.62 microg/L range. Sewage and stabilized sludge samples contained mean PAH concentrations, as sum of 16 compounds, in the concentration range of 1.44-1.26 mg/kg, respectively. Extraction and clean up procedures for sludge samples were validated using EPA certified reference material IRM-104 (CRM No. 912). Instrumental analyses were performed by coupling HPLC with UV-diode array detection (UV-DAD) and fluorescence detection (FLD).     

Separation of purine and pyrimidine bases by ion chromatography with direct conductivity detection

A simple, rapid and accurate method for the simultaneous determination of four purine and pyrimidine bases (cytosine, 5-methylcytosine, adenine and N6-methyladenine) has been developed. The quantitative determination of these bases was accomplished by ion chromatography (IC) with direct conductivity detection (CD) based on their ionization in acidic medium without chemical suppression. The recovery of cytosine, 5-methylcytosine, and adenine in calf thymus DNA was more than 98% (n=3) and the relative standard deviation (RSD, n=5) less than 2.4%. In a single chromatographic run, the four bases could be separated and determined in less than 10 min. The detection limits were found to be 0.05 microg/mL for cytosine, 0.08 microg/mL for 5-methylcytosine, 0.07 microg/mL for adenine, and 0.07 microg/mL for N6-methyladenine. Linear ranges were 0.2-95.1 microg/mL for cytosine (r2=0.9996), 0.3-196.6 microg/mL for 5-methylcytosine (r2=0.9994), 0.3-105.5 microg/mL for adenine (r2=0.9998), and 0.3-159.1 microg/mL for N6-methyladenine (r2=0.9999). With the proposed method, purine and pyrimidine bases could be successfully detected in calf thymus DNA. We also determined these bases in calf thymus DNA using RP-HPLC. Compared to RP-HPLC, the IC method offers advantages such as high selectivity and simple mobile phase.     

Rapid and simple determination of selenium in blood serum by inductively coupled plasma–mass spectrometry (ICP–MS)

An inductively coupled plasma mass spectrometer (ICP-MS) with a rapid sample-preparative procedure was used for the determination of selenium in blood serum. Blood serum was prepared by dilution in an acidic solution consisting of nitric acid (1%), X-triton (0.1%) and 1-butanol (0.8%). A calibration curve was established for 1-40 microg mL(-1) (r(2)>0.99). The limit of detection was 0.5 microg mL(-1). Repeatability and intermediate precision were satisfactory with relative standard deviations (RSD) of 2.0% and 3.2%, respectively. This method was easily applied to reference materials with satisfactory accuracy. Good correlation (r(2)=0.96) was observed between ICP-MS and atomic absorption spectrometry (AAS) for the determination of (82)Se in blood serum from 23 patients. These results suggest that the sample preparative procedure coupled with ICP-MS can be used for the routine determination of (82)Se in human blood serum.