Determination of the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human hair by solid-phase extraction and gas chromatography-mass spectrometry

Summary A method for the determination of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human hair has been developed and validated. Hair samples (200 mg) were dissolved in NaOH (1 M) and PhIP was isolated by successive solid-phase extraction on a polystyrene-divinylbenzene column and on a silica-based mixed-mode column with C₈ and-SO₃ ⁻ functional groups. Quantification was performed by gas chromatography-electron-impact ionization high-resolution mass spectrometry in selected-ion-monitoring mode. The method was validated for determination of PhIP in the concentration range 0.5–25 ng g⁻¹ hair with [²H₃]PhIP as internal standard. The limit of quantification was 0.26 ng g⁻¹ hair. Within-day and between-day precision were in the ranges 1–27% and 2–15% relative standard deviation, respectively. The hair sample used for method validation was found to contain 0.26 ng PhIP g⁻¹ hair.     

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

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

Simultaneous RP-LC Determination of Losartan Potassium, Ramipril, and Hydrochlorothiazide in Pharmaceutical Preparations

A simple, rapid, and precise reversed-phase high-performance liquid chromatographic method has been developed for simultaneous determination of losartan potassium, ramipril, and hydrochlorothiazide. The three drugs were separated on a 150 mm × 4.6 mm i.d., 5 μm particle, Cosmosil C₁₈ column. The mobile phase was 0.025 m sodium perchlorate–acetonitrile, 62:38 (v/v), containing 0.1% heptanesulphonic acid, pH adjusted to 2.85 with orthophosphoric acid, at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 215 nm. The method was validated for linearity, accuracy, precision, and limit of quantitation. Linearity, accuracy, and precision were acceptable in the ranges 35–65 μg mL⁻¹ for losartan, 1.75–3.25 μg mL⁻¹ for ramipril, and 8.75–16.25 μg mL⁻¹ for hydrochlorothiazide.     

Simple, Sensitive, and Rapid LC–ESI-MS Method for Quantification of Mitiglinide in Human Urine

A sensitive and specific liquid chromatographic method with electrospray ionization mass spectrometry (LC–ESI-MS) has been developed and validated for identification and quantification of mitiglinide in human urine. A simple liquid–liquid extraction procedure was followed by separation on a C₁₈ column with gradient elution, and detection using a single-quadrupole mass spectrometer in selected-ion-monitoring (SIM) mode. The method was tested using six different batches of urine. Linearity was established for the mitiglinide concentrations in the range 0.005–1.0 μg mL⁻¹, with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. Intra- and inter-day precision (as RSD, %) was below 10% and accuracy for mitiglinide ranged from 85 to 115%. The lower limit of quantification was reproducible at 0.002 μg mL⁻¹ for 500 μL urine. The proposed method enables unambiguous identification and quantification of mitiglinide in pre-clinical and clinical studies.     

Development of a liquid chromatography-mass spectrometry method for the determination of ursolic acid in rat plasma and tissue: application to the pharmacokinetic and tissue distribution study

A fast and sensitive liquid chromatography–mass spectrometry method was developed for the determination of ursolic acid (UA) in rat plasma and tissues. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was performed on a 3.5 μm Zorbax SB-C18 column (30 mm × 2.1 mm) with a mobile phase consisting of methanol and aqueous 10 mM ammonium acetate using gradient elution. Quantification was performed by selected ion monitoring with (m/z)⁻ 455 for UA and (m/z)⁻ 469 for the IS. The method was validated in the concentration range of 2.5 − 1470 ng mL⁻¹ for plasma samples and 20 − 11760 ng g⁻¹ for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 1.6% to 7.1% and 3.7% to 9.0%, respectively, and the intra- and inter-day assay accuracy was 84.2 − 106.9% and 82.1 − 108.1%, respectively. Recoveries in plasma and tissues ranged from 83.2% to 106.2%. The limits of detections were 0.5 ng mL⁻¹ or 4.0 ng g⁻¹. The recoveries for all samples were >90%, except for liver, which indicated that ursolic acid may metabolize in liver. The main pharmacokinetic parameters obtained were T _max = 0.42 ± 0.11 h, C _max = 1.10 ± 0.31 μg mL⁻¹, AUC = 1.45 ± 0.21 μg h mL⁻¹ and K _a = 5.64 ± 1.89 h⁻¹. The concentrations of UA in rat lung, spleen, liver, heart, and cerebellum were studied for the first time. This method is validated and could be applicable to the investigation of the pharmacokinetics and tissue distribution of UA in rats.     

A sensitive liquid chromatographic-mass spectrometric method for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma

A liquid chromatographic–mass spectrometric (LC–MS) method has been developed and validated for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma. After addition of the internal standard, domperidone, plasma samples were extracted by liquid–liquid extraction with ethyl acetate and separated on an Apollo C₁₈ column (250 mm × 4.6 mm, 5 μm), with methanol–0.01% formic acid water (60:40, v/v) as mobile phase, within a runtime of 12.0 min. The analytes were detected without interference in the selected ion monitoring (SIM) mode with positive electrospray ionization. The linear range was 1.0–500 ng mL⁻¹ for dehydroevodiamine and 2.0–1,000 ng mL⁻¹ for limonin, with lower limits of quantitation of 1.0 and 2.0 ng mL⁻¹, respectively. Intra-day and inter-day precision were within 6.0% and 10.9%, respectively, for both analytes, and the accuracy (relative error, RE, %) was less than 4.8% and 6.5%, respectively. The validated method was successfully applied to a comparative pharmacokinetic study of dehydroevodiamine and limonin in rat plasma after oral administration of dehydroevodiamine, limonin, and an aqueous extract of Evodiae fructus. The results indicated there were obvious differences between the pharmacokinetic behavior after oral administration of an aqueous extract of Evodiae fructus compared with single substances.     

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

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

     

Determination of organic micro-pollutants such as personal care products, plasticizers and flame retardants in sludge

In this study, a method for the determination of organic micro-pollutants, i.e. personal care products such as synthetic musk fragrances, household bactericides, organophosphate flame retardants and plasticizers, as well as phthalates in sludge, has been developed. This method is based on lyophilisation and accelerated solvent extraction followed by clean-up steps, i.e. solid phase extraction and size exclusion chromatography. The determination is performed by gas chromatography coupled to mass spectrometry. Stable isotope-labelled compounds such as musk xylene (MX D₁₅), tri-n-butylphosphate (TnBP D₂₇) and triphenylphosphate (TPP D₁₅) were used as internal standards. Recovery rates were determined to be 36–114% (with typical relative standard deviation of 5% to 23%) for the target compounds. The limit of detection was 3–30 ng g⁻¹, and the limit of quantification was 10–100 ng g⁻¹ dry matter.     

Simultaneous Determination of Rosiglitazone and Metformin in Pharmaceutical Preparations by LC

In the present study, a novel, fast and simple liquid chromatographic method was developed and validated for the simultaneous determination of rosiglitazone and metformin in pharmaceutical preparations. The separation was achieved on a phenyl column (250 × 4.6 mm i.d., 5 μm) using a mobile phase composed of acetonitrile:10.0 mM phosphate buffer pH 5.5 (70:30, v/v). The flow rate was 1 mL min⁻¹. UV detection was performed at 245 nm and verapamil was used as internal standard. The developed method was validated in terms of stability, specificity, sensitivity, linearity, accuracy, precision and robustness. The limit of quantification was 0.02 μg mL⁻¹ for both drugs. The method developed was successfully applied to the simultaneous determination of rosiglitazone and metformin in pharmaceutical preparations. The results were compared to two methods reported in the literature and no significant difference was found statistically.     

Hapten synthesis and enzyme-linked immunosorbent assay for phosmet residues: assay optimization and investigation of matrix effects from different food samples

In this study, a panel of haptens was synthesized for immunoconjugate preparation, and several haptens for heterologous tracer conjugates were also prepared. A highly sensitive polyclonal antibody against the organophosphorus insecticide phosmet was obtained and competitive direct enzyme-linked immunosorbent assays (cd-ELISA) for this pesticide were developed. In the cd-ELISA, the limit of detection (IC₁₅) was 0.6 μg kg⁻¹ and the sensitivity (IC₅₀) was 20 μg kg⁻¹. The suitability of the ELISA for pesticide quantification in peach, apple, orange juice, and apple juice was also studied. Good accuracy and precision were obtained with mean recoveries between 78% and 102.3% and mean coefficients of variation below 13.63%. Validation of the ELISA was conducted by high-performance liquid chromatography. The correlation between the data obtained using the microwell assay and the high-performance liquid chromatography was good (R ² = 0.9849). The developed immunoassay methods were suitable for the rapid quantitative or qualitative determination of phosmet in food samples.     

Multisyringe flow injection spectrophotometric determination of uranium in water samples

A multisyringe flow injection analysis method for the determination of uranium in water samples was developed. The methodology was based on the complexation reaction of uranium with arsenazo (III) at pH 2.0. Uranium concentrations were spectrophotometrically detected at 649 nm using a light emitting diode. Under the optimized conditions, a linear dynamic range from 0.1 to 4.0 μg mL⁻¹, a 3σ detection limit of 0.04 μg mL⁻¹, and a 10σ quantification limit of 0.10 μg mL⁻¹ were obtained. The reproducibility (%) at 0.5, 2.5, and 4.0 μg mL⁻¹ was 2.5, 0.9, and 0.6%, respectively (n = 10). The interference effect of some ions was tested. The proposed method was successfully applied to the determination of uranium in water samples.     

Organosilica composite for preconcentration of phenolic compounds from aqueous solutions

A new adsorbent is proposed for the solid-phase extraction of phenol and 1-naphthol from polluted water. The adsorbent (TX-SiO₂) is an organosilica composite made from a bifunctional immobilized layer comprising a major fraction (91%) of hydrophilic diol groups and minor fraction (9%) of the amphiphilic long-chain nonionic surfactant Triton X-100 (polyoxyethylated isooctylphenol) (TX). Under static conditions phenol was quantitatively extracted onto TX-SiO₂ in the form of a 4-nitrophenylazophenolate ion associate with cetyltrimethylammonium bromide. The capacity of TX-SiO₂ for phenol is 2.4 mg g⁻¹ with distribution coefficients up to 3.4 × 10⁴ mL g⁻¹; corresponding data for 1-naphthol are 1.5 mg g⁻¹ and 3 × 10³ mL g⁻¹. The distribution coefficient does not change significantly for solution volumes of 0.025–0.5 L and adsorbent mass less than 0.03 g; 1–90 μg analyte can be easily eluted by 1–3 mL acetonitrile with an overall recovery of 98.2% and 78.3% for phenol and 1-naphthol, respectively. Linear correlation between acetonitrile solution absorbance (A ₅₄₀) and phenol concentration (C) in water was found according to the equation A ₅₄₀ = (6 ± 1) × 10⁻² + (0.9 ± 0.1)C (μmol L⁻¹) with a detection range from 1 × 10⁻⁸ mol L⁻¹ (0.9 μL g⁻¹) to 2 × 10⁻⁷ mol L⁻¹ (19 μL g⁻¹), a limit of quantification of 1 μL g⁻¹ (preconcentration factor 125), correlation coefficient of 0.936, and relative standard deviation of 2.5%. A solid-phase colorimetric method was developed for quantitative determination of 1-naphthol on adsorbent phase using scanner technology and RGB numerical analysis. The detection limit of 1-naphthol with this method is 6 μL g⁻¹ while the quantification limit is 20 μL g⁻¹. A test system was developed for naked eye monitoring of 1-naphthol impurities in water. The proposed test kit allows one to observe changes in the adsorbent color when 1-naphthol concentration in water is 0.08–3.2 mL g⁻¹.     

Development of a Validated Stability-Indicating LC Assay and Stress Degradation Studies of Linezolid in Tablets

This paper describes the validation of an isocratic LC method for the assay of linezolid in tablets. Validation parameters such as linearity, precision, accuracy, specificity, limit of detection, limit of quantitation and robustness were determined. LC was carried out by reversed phase technique on an RP-18 column with a mobile phase composed of 1% acetic acid:methanol:acetonitrile (50:25:25, v/v/v). Linezolid and your combination drug product were exposed to acid, base, oxidation, dry heat and photolytic stress conditions. A linear response (r > 0.9999) was observed in the range of 8.0–20.0 μg mL⁻¹. The retention time of linezolid was 4.6 min. The method showed good recoveries and intra- and inter-day relative standard deviations were less than 1.0%. The LOD and LOQ were 0.21 and 0.63 μg mL⁻¹, respectively. The developed LC method for determination of related substances and assay determination of linezolid can be used to evaluate the quality of regular production samples. It can also be used to test the stability samples of linezolid.     

LC-DAD Determination of Fleroxacin in Bulk and Pharmaceutical Dosage Forms

Fleroxacin is a third generation fluoroquinolone with broad spectrum antibacterial activity. In this work an LC-DAD method for the analysis of fleroxacin was developed and validated using UV detection at 286 nm. The method was validated for linearity, precision, robustness, LOD, LOQ, specificity and accuracy at concentrations of 0.2–20.0 μg mL⁻¹ and r ² = 1. The LOD and LOQ were 0.059 and 0.197 μg, respectively, the recoveries were 99.92–102.0% and the CV was less than 2.0%. The LC-DAD validated method provided analytical sensitivity, specificity and reproducibility suitable for quality control analysis.     

Determination of total Sb,Se, Te,and Bi and evaluation of their inorganic species in garlic by hydride-generation–atomic-fluorescence spectrometry

A sensitive and simple analytical method has been developed for determination of Sb(III), Sb(V), Se(IV), Se(VI), Te(IV), Te(VI), and Bi(III) in garlic samples by using hydride-generation–atomic-fluorescence spectrometry (HG–AFS). The method is based on a single extraction of the inorganic species by sonication at room temperature with 1 mol L⁻¹ H₂SO₄ and washing of the solid phase with 0.1% (w/v) EDTA, followed by measurement of the corresponding hydrides generated under two different experimental conditions directly and after a pre-reduction step. The limit of detection of the method was 0.7 ng g⁻¹ for Sb(III), 1.0 ng g⁻¹ for Sb(V), 1.3 ng g⁻¹ for Se(IV), 1.0 ng g⁻¹ for Se(VI), 1.1 ng g⁻¹ for Te(IV), 0.5 ng g⁻¹ for Te(VI), and 0.9 ng g⁻¹ for Bi(III), in all cases expressed in terms of sample dry weight.     

LC Method for Studies on the Stability of Sibutramine in Soft Gelatin Capsules

A sensitive and rapid liquid chromatographic method was successfully developed and validated for the determination of sibutramine hydrochloride in bulk and capsules. Sibutramine in the presence of its degradation products was analyzed using UV detection at 225 nm. Chromatography was performed on a reversed-phase C₈ (150 × 4.0 mm I.D., 5 μm) analytical column under isocratic conditions. The mobile phase was composed of acetonitrile:water (aqueous phase containing 0.3% triethylamine and pH adjusted to 7.0) (75:25, v/v) at a flow-rate of 1.1 mL min⁻¹. No chromatographic interference was found during the analysis. Light was the stress condition which most contributed to sibutramine degradation. The method showed a linear response (r > 0.999) from 30 to 90 μg mL⁻¹. The mean recovery for capsules was 101.2%. Inter-day assays showed relative standard deviations of 0.42 and 1.62% for bulk and capsules, respectively. The developed method is able to separate sibutramine from its major degradation products and it may be used in the quality control of this active pharmaceutical ingredient in both bulk and capsules.     

Kinetic determination of silver at trace level based on its catalytic effect on a ligand substitution reaction

It is observed that Ag(I) catalyzes the rate of substitution of phenylhydrazine (PhNHNH₂) into hexacyanoferrate(II), producing a cherry red colored complex, [Fe(CN)₅PhNHNH₂]³⁻. The reaction was monitored at 488 nm leading to the formation of the complex under the conditions: [Fe(CN)₆]⁴⁻ (5.0 × 10⁻³ mol dm⁻³), PhNHNH₂ (2.0 × 10⁻³ mol dm⁻³), temperature (25 ± 0.1 °C), pH (2.8 ± 0.02), and ionic strength, I (0.02 mol dm⁻³), (KNO₃). Under optimum conditions, absorbance at fixed times (A _t ) is linearly related to Ag(I) in the concentration range 10.79–97.08 ng cm⁻³, in the presence of several diverse ions. The highest percentage error and relative standard deviations in the entire range of Ag(I) determination are found to be 2.5% and 0.16, with a detection limit of 8.75 ng cm⁻³ of silver(I). The experimental accuracies expressed in terms of percentage recoveries are in the range of 97.87–102.50. The method was successfully applied for the determination of Ag(I) in a few synthetic samples and found to be in good agreement with those obtained from atomic absorption spectrophotometry (AAS). The validity of the proposed method has also been tested for Ag(I) determination in spiked drinking water samples. The present catalytic kinetic method (CKM) is highly sensitive, selective, reproducible, and inexpensive. A review of recently published catalytic spectrophotometric methods for determination of Ag(I) has also been presented for comparison.     

Determination and Validation of an LC Method for Fluvoxamine in Tablets

A new, simple, rapid and specific reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for the determination of fluvoxamine in pharmaceutical dosage forms. The HPLC separation was achieved on a C₁₈ μ-Bondapack column (250 mm × 4.6 mm) using a mobile phase of acetonitrile–water (80:20, v/v) at a flow rate of 1 mL min⁻¹. Proposed method is based on the derivatization of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) in borate buffer of pH 8.5 to yield a orange product. The HPLC method is based on measurement of the derivatized product using UV-visible absorbance detection at 450 nm. The method was validated for specificity, linearity, precision, accuracy, robustness. The degree of linearity of the calibration curves, the percent recoveries of fluvoxamine, the limit of detection and quantification, for the HPLC method were determined. The assay was linear over the concentration range of 45–145 ng mL⁻¹ (r = 0.9999). Limit of detection and quantification for fluvoxamine were 15 and 50 ng mL⁻¹, respectively. The results of the developed procedure (proposed method) for fluvoxamine content in tablets were compared with those by the official method. The method was found to be simple, specific, precise, accurate, reproducible and robust.     

Analysis of Spectinomycin by HPLC with Evaporative Light-Scattering Detection

A high-performance liquid chromatographic method with evaporative light-scattering detection (ELSD) has been developed for analysis of spectinomycin and related impurities. Separation of spectinomycin from structurally related impurities was achieved on a C₁₈ column. The optimized mobile phase was 25 mmol L⁻¹ ammonium acetate (pH 7.5)-methanol, 90:10 (v/v), at a flow rate of 0.6 mL min⁻¹. The temperature of the drift tube of the ELSD was 95°C and the flow rate of carrier gas was 2.2 L min⁻¹. The accuracy, specificity, precision, linearity, sensitivity, and robustness of the method were validated in accordance with ICH guidelines. In addition to determination of spectinomycin and related impurities, the method is also ideal for determination of the salts spectinomycin hydrochloride and spectinomycin sulfate.     

Determination of pheniramine enantiomers in eye drop by capillary electrophoresis using hydroxypropyl-β-cyclodextrin as chiral selector

A capillary zone electrophoresis procedure has been developed for the chiral determination of pheniramine in eye drop. Native and derivative cyclodextrins (CDs) including γ-CD, β-CD, hydroxypropyl-β-CD and dimethyl-β-CD were tested as chiral selectors. Using 30 mM hydroxypropyl-β-CD in 50 mM phosphate buffer (pH 3.0), the acceptable resolution value (R = 1.55) was obtained. The assay was validated for linearity (3.3 × 10⁻⁶–5.0 × 10⁻⁴ M; R ² = 0.9996), limit of detection (3.3 × 10⁻⁶ M), limit of quantification (8.5 × 10⁻⁶ M), analytical precision by terms of intra- and inter-day variability (RSD ≤ 2.57%), and accuracy (recovery ≥ 89.3%). The content of pheniramine in eye drop obtained by the proposed method was in good agreement with the declared value. The results indicated that pheniramine in the eye drop was present as the racemate.