Development and validation of an analytical method for the quantification of cytochrome c in skin transport studies

A simple isocratic HPLC method for the quantification of Cytochrome c in skin permeation samples was developed and validated. The mobile phase comprised a 41 : 59 mixture of an organic phase A (0.1% trifluoroacetic acid in a 90 : 10 mixture of MeCN–H₂O) and an aqueous phase B (0.1% trifluoroacetic acid in H₂O). The Cytochrome c retention and run times were 2.62 and 8.0 min, respectively—much shorter than those for existing gradient methods. The response was accurate, precise and linear from 2.5 to 25 μg/mL. The mean recoveries for intra‐day and inter‐day analysis ranged from 88.5 to 103.8% and the RSD varied from 0.05 to 1.55%. The assay was used to quantify transport of Cytochrome c across intact and laser‐microporated porcine skin in vitro. Cytochrome c permeation and the amount of protein retained within the membrane over 24 h were quantified as a function of the number of micropores. Although no Cytochrome c permeation was observed across intact skin, laser microporation enabled delivery of 22.9 ± 3.3 and 56.0 ± 15.9 μg/cm² of the protein across skin samples with 300 and 1800 micropores, respectively. In conclusion, the HPLC method provided a fast, efficient means to quantify Cytochrome c in samples from skin transport studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a high-performance liquid chromatography method for quantification of egonol and homoegonol in Styrax species

Styrax camporum Pohl, known in Brazil as 'estoraque do campo' or 'cuia de brejo', has been used in the treatment of gastrointestinal diseases. The therapeutic action of S. camporum has been attributed to the ethyl acetate fraction, although the chemical composition of this fraction has not yet been analyzed. In this study, a high-performance liquid chromatography photodiode array detection (HPLC-PAD) method for analysis of Brazilian Styrax species has been developed. The compounds egonol (1) and homoegonol (2) were found to be present in all the samples investigated by HPLC. These compounds were isolated by open column chromatography followed by preparative TLC, and were identified by 1H NMR. Compounds 1 and 2 were thus proposed as phytochemical markers for Styrax, owing to their biological properties and presence in other Styrax species. The developed method has been validated and successfully applied for quantification of 1 and 2 in S. camporum dried leaves and crude ethanolic extracts from S. ferrugineus and S. pohlii aerial parts.     

Development and validation of HPLC method for imiquimod determination in skin penetration studies

A simple, rapid and sensitive analytical procedure for the measurement of imiquimod in skin samples after in vitro penetration studies has been developed and validated. In vitro penetration studies were carried out in Franz diffusion cells with porcine skin. Tape stripping technique was used to separate the stratum corneum (SC) from the viable epidermis and dermis. Imiquimod was extracted from skin samples using a 7:3 (v/v) methanol:acetate buffer (100 mM, pH 4.0) solution and ultrasonication. Imiquimod was analyzed by HPLC using C(8) column and UV detection at 242 nm. The mobile phase used was acetonitrile:acetate buffer (pH 4.0, 100 mM):diethylamine (30:69.85:0.15, v/v) with flow rate 1 mL/min. Imiquimod eluted at 4.1 min and the running time was limited to 6.0 min. The procedure was linear across the following concentration ranges: 100-2500 ng/mL for both SC and tape-stripped skin and 20-800 ng/mL for receptor solution. Intra-day and inter-day accuracy and precision values were lower than 20% at the limit of quantitation. The recovery values ranged from 80 to 100%. The method is adequate to assay imiquimod from skin samples, enabling the determination of the cutaneous penetration profile of imiquimod by in vitro studies.     

Development and validation of a rapid high-performance liquid chromatography method for the quantification of exenatide

The objective was to develop a simple HPLC method to quantify exenatide—a 39 amino acid residue incretin mimetic used in diabetes therapy. To date, only non‐validated, sometimes incomplete, gradient methods have been reported in the literature. Isocratic separation was achieved using a C₄ column and a mixed solvent system, A–B–C (48:45:7, v/v/v; pH* 5.2), where A represents KH₂PO₄ (pH 4.5; 0.1 m ) and MeCN (60:40, v/v), B corresponds to NaClO₄·H₂O (pH 6.0; 0.2 m ) and MeCN (60:40, v/v), and C is water. Exenatide eluted at 3.64 min and the total run time was 6 min. The method was specific and the response was accurate, precise and linear from 0.75 to 25 µg/mL. It was used to quantify exenatide transport across intact and laser‐porated porcine skin in vitro as a function of laser fluence [0 (i.e. intact skin), 9 and 15 J/cm², respectively]. Although no permeation was observed using intact skin, cumulative exenatide permeation after 8 h through laser porated skin was 9.6 ± 6.5 and 12.4 ± 6.4 µg/cm² at fluences of 9 and 15 J/cm², respectively. This is the first validated isocratic method for exenatide quantification and it may be of use in quality control analysis and with other biological matrices. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of HPLC method for simultaneous estimation of piperine and guggulsterones in compound Unani formulation (tablets) and a nanoreservoir system

An attempt has been made to develop and validate a simultaneous HPLC method for novel approach of drug release via oil‐in‐water (o/w) nanoemulsion formulation and Habb‐e‐Khardal Unani tablet containing piperine and guggul sterones E and Z as main ingredients. Nanoemulsion was prepared by titration method using sefsol‐218 as an oily phase, cremophor‐EL as a surfactant, transcutol as a co‐surfactant and distilled water as an aqueous phase. The formulation was optimized on the basis of thermodynamic stability and dispersibilty test. The nanoformulation was evaluated for particle size, surface morphology, electrical conductivity and viscosity determination. The in vitro dissolution was carried out by dialysis bag method. Drugs were quantified using an HPLC method developed in‐house with a C₁₈ column as stationary phase and acetonitrile and water as mobile phase at λ_max of 240 nm. The optimized formulation showed higher drug release, lower droplet size and less viscosity as compared with the conventional Habb‐e‐Khardal Unani tablet. The present study illustrated the potential of nanoemulsion dosage form in improving biopharmaceutic performance of piperine and guggul sterone. The HPLC method was also found to be quite sufficient for the routine quality control of formulations containing piperine and guggul sterone E and Z as ingredients and also for in vitro drug release studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of a rapid and sensitive HPLC method for the quantification of 5‐fluorocytosine and its metabolites

To study the intracellular metabolism of the prodrug 5‐fluorocytosine (5FC), we developed a novel reverse‐phase high‐performance liquid chromatography method to simultaneously detect 5FC and its four major anabolic metabolites: 5‐fluorouracil, 5‐fluorouridine, 5‐fluorouridine‐monophosphate and 5‐fluoro‐2′deoxyuridine‐5′‐monophosphate. Separation of each compound was accomplished under isocratic conditions using a C₁₈ column and mobile phase of formic acid–water (1 : 99 v/v). The method was validated for both accuracy and reproducibility in cell culture media. Additionally, metabolites were assessed for stability at ambient temperatures and following freeze–thaw cycles. Calibration curves were linear over a range of 1–200 μg/mL. Limit of quantification for four of the five compounds was 1 μg/mL in cell culture media (RSD < 11%). This method was successfully used to monitor intracellular conversion of 5FC to its metabolic products over a 24h period. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of acetyl-CoA and malonyl-CoA in animal tissues

A highly sensitive and specific LC‐MS/MS method was developed for simultaneous estimation of acetyl co‐enzyme A (ACoA) and malonyl co‐enzyme A (MCoA) in surrogate matrix using n‐propionyl co‐enzyme A as an internal standard (IS). LC‐MS/MS was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. Simple acidification followed by dilution using an assay buffer process was used to extract ACoA, MCoA and IS from surrogate matrix and tissue samples. The total run time was 3 min and the elution of both analytes (ACoA, MCoA) and IS occurred at 1.28 min; this was achieved with a mobile phase consisting of 5 mM ammonium formate (pH 7.5)–acetonitrile (30:70, v/v) delivered at a flow rate of 1 mL/min on a monolithic RP‐18e column. A linear response function was established for the range of concentrations 1.09–2187 and 1.09–2193 ng/mL for ACoA and MCoA, respectively. The intra‐ and inter‐day precision values for ACoA and MCoA met the acceptance as per FDA guidelines. ACoA and MCoA were stable in a battery of stability studies viz. bench‐top, auto‐sampler and long‐term. The developed assay was used to quantitate ACoA and MCoA levels in various tissues of rat. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method for quantification of Δ9‐tetrahydrocannabinolic acid A (THCA‐A), THC,CBN and CBD in hair

For analysis of hair samples derived from a pilot study (‘in vivo’ contamination of hair by sidestream marijuana smoke), an LC‐MS/MS method was developed and validated for the simultaneous quantification of Δ9‐tetrahydrocannabinolic acid A (THCA‐A), Δ9‐tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD). Hair samples were extracted in methanol for 4 h under occasional shaking at room temperature, after adding THC‐D₃, CBN‐D₃, CBD‐D₃ and THCA‐A‐D₃ as an in‐house synthesized internal standard. The analytes were separated by gradient elution on a Luna C18 column using 0.1% HCOOH and ACN + 0.1% HCOOH. Data acquisition was performed on a QTrap 4000 in electrospray ionization‐multi reaction monitoring mode. Validation was carried out according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Limit of detection and lower limit of quantification were 2.5 pg/mg for THCA‐A and 20 pg/mg for THC, CBN and CBD. A linear calibration model was applicable for all analytes over a range of 2.5 pg/mg or 20 pg/mg to 1000 pg/mg, using a weighting factor 1/x. Selectivity was shown for 12 blank hair samples from different sources. Accuracy and precision data were within the required limits for all analytes (bias between ?0.2% and 6.4%, RSD between 3.7% and 11.5%). The dried hair extracts were stable over a time period of one to five days in the dark at room temperature. Processed sample stability (maximum decrease of analyte peak area below 25%) was considerably enhanced by adding 0.25% lecithin (w/v) in ACN + 0.1% HCOOH for reconstitution. Extraction efficiency for CBD was generally very low using methanol extraction. Hence, for effective extraction of CBD alkaline hydrolysis is recommended. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a UPLC method for quantification of antiviral agent,Acyclovir in lipid-based formulations

PurposeThe objective of the current study is to evaluate the Ultra Performance Liquid Chromatography (UPLC) method for quantification of Acyclovir in lipid-based formulations.MethodA simple, rapid, reliable and precise reversed phase UPLC method has been developed and validated according to the regulatory guidelines, which composed of isocratic mobile phase; 0.25% formic acid (FA) in Milli-Q water with a flow rate of 0.5 ml/min, and column BEH C18 (2.1 × 50 mm, 1.7 μm). The detection was carried out at 254 nm.ResultsThe developed UPLC method was found to be rapid (1.2 min run time), selective with well resoluted Acyclovir peak (0.89 min) from different lipid matrices and sensitive (Limit of Detection (LOD) was 0.3 ppm and Lower Limit of Quantification (LLOQ) was 1 ppm). The accuracy and precision were determined and were perfectly matching with the standard FDA limits.ConclusionThe study showed that the proposed UPLC method can be used for the assessment of drug purity, stability, solubility and lipid-formulation release profile with no interference of excipients or related substances of active pharmaceutical ingredient.     

Development and validation of a high‐resolution LTQ Orbitrap MS method for the quantification of isoflavones in wastewater effluent

Isoflavones and coumestranes are the most important classes of compounds among phytoestrogens; by binding to estrogen receptors, they mimic or modulate the effect on the endogenous receptors. Little information can be found in literature about the presence of isoflavones and coumestrol in the environment, even if it is known that this may have significance, being these substances classified as endocrine disrupting compounds. In this research, we aim to explore the capabilities of the LTQ Orbitrap Discovery hybrid MS in full‐scan acquisition mode, with high resolution, to validate an analytical method for the quantification of nine isoflavones (genistein, genistin, glycitein, daidzein, daidzin, (R,S)‐equol, biochanin A, formononetin and coumestrol) in wastewater samples. The correlation coefficients of calibration curves of the nine analyzed compounds were in a range of 0.996–0.999; recoveries at two different levels of concentration (0.05 and 0.5 µg/l) were in the range 73–98%, and the limits of detection ranged between 0.0014 and 0.017 µg/l, proving that this method is sensitive enough in comparison with other methods available in literature. This method has been applied for the analysis of 20 wastewater treatment plants in County Cork, Ireland. Copyright © 2015 John Wiley & Sons, Ltd.     

Validated HPLC method for the standardization of Phyllanthus niruri (herb and commercial extracts) using corilagin as a phytochemical marker

Phyllanthus niruri L., commonly known in Brazil as ‘quebra‐pedra’, has long been used in the treatment of diverse diseases and especially urolithiasis. The therapeutic effects of P. niruri are attributed to various compounds present in the plant, including the hydrolysable tannin corilagin. In the present study, high‐performance liquid chromatography (HPLC‐/PAD) profiles of leaves and commercial extracts of P. niruri were examined and three compounds, found to be present in all of the samples studied, were isolated by open column chromatography over C₁₈ silica gel followed by preparative HPLC. These compounds were identified by nuclear magnetic resonance as corilagin, rutin and ethyl 3,4,5‐trihydroxybenzoate. Corilagin, which has been proposed as a phytochemical marker for P. niruri, was employed as an external standard in the development and validation of a rapid and efficient qualitative and quantitative HPLC assay for the analyte. The method may be applied in the standardization of herbs and phytomedicines commercialized in Brazil as quebra‐pedra. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a liquid chromatography/tandem mass spectrometry method for the quantification of flucloxacillin and cloxacillin in microdialysis samples

In the present study we developed and validated a liquid chromatography/tandem mass spectrometry (LC‐MS/MS) assay for the determination of flucloxacillin in human plasma and microdialysis samples and cloxacillin in microdialysis samples, using oxacillin as the internal standard for the assay. The samples were separated on a UPLC BEH C₁₈,1.7 µm column (2.1 × 50 mm) and analyzed by a tandem–quadrupole mass spectrometer in multiple reaction monitoring mode using an electronspray ionization interface. For flucloxacillin the method was demonstrated to be accurate and precise in the linearity range of 1–30 mg/L in plasma and 0.05–5.0 mg/L for microdialysate with a regression coefficient (r) of 0.9986 and 0.9989 in plasma and microdialysate respectively. For cloxacillin it was accurate and precise in the range of 0.1–5.0 mg/L for microdialysate with a regression coefficient of 0.9972. The method presents a high sensitivity for flucloxacillin (lower limit of quantification of 1 mg/L for plasma and 0.05 mg/L for microdialysis samples) combined with a low within‐ and between‐day variation (<5.0% for flucloxacillin and cloxacillin in microdialysis samples and <6.5% for plasma samples of flucloxacillin). The validation experiments for the microdialysis probes showed a relative recovery of 85.5% for flucloxacillin at a flow rate of 1.0 μL/min. The results justify the use of this assay for clinical studies for measuring free unbound tissue concentrations of flucloxacillin in patients with a Staphylococcus aureus bacteremia. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a quantification method for ziyuglycoside I and II in rat plasma: Application to their pharmacokinetic studies

This study provided a novel and generally applicable method to determine ziyuglycoside I and ziyuglycoside II in rat plasma based on liquid chromatography with tandem mass spectrometry. A single step of liquid–liquid extraction with n‐butanol was utilized, and ginsenoside Rg3 was chosen as internal standard. Final extracts were analyzed based on liquid chromatography with tandem mass spectrometry. Chromatographic separation was achieved using a Thermo Golden C₁₈ column, and the applied gradient elution program allowed for the simultaneous determination of two ziyuglycosides in a one‐step chromatographic separation with a total run time of 10 min. The fully validated methodology for both analytes demonstrated high sensitivity (the lower limit of quantitation was 2.0 ng/mL), good accuracy (% RE ≤ ± 15) and precision (% RSD ≤ 15). The average recoveries of both ziyuglycosides and internal standard were all above 75% and no obvious matrix effect was found. This method was then successfully applied to the preclinical pharmacokinetic studies of ziyuglycoside I and ziyuglycoside II. The presently developed methodology would be useful for the preclinical and clinical pharmacokinetic studies for ziyuglycoside I and ziyuglycoside II.     

Development and validation of a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for the quantification of tigecycline in rat brain tissues

Tigecycline (TIG), a derivative of minocycline, is the first in the novel class of glycylcyclines and is currently indicated for the treatment of complicated skin structure and intra‐abdominal infections. A selective, accurate and reversed‐phase high‐performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) method was developed for the determination of TIG in rat brain tissues. Sample preparation was based on protein precipitation and solid phase extraction using Supel‐Select HLB (30 mg/1 mL) cartridges. The samples were separated on a YMC Triart C₁₈ column (150 mm x 3.0 mm. 3.0 µm) using gradient elution. Positive electrospray ionization (ESI+) was used for the detection mechanism with the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range of 150–1200 ng/mL for rat brain tissue. The precision and accuracy for all brain analyses were within the acceptable limit. The mean extraction recovery in rat brain was 83.6%. This validated method was successfully applied to a pharmacokinetic study in female Sprague Dawley rats, which were given a dose of 25 mg/kg TIG intraperitoneally at various time‐points. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of a UHPLC‐qTOF‐MS method for quantification of fuziline in rat plasma and its application in a pharmacokinetic study

A specific and sensitive UHPLC‐qTOF‐MS method was developed and validated for quantification of fuziline in rat plasma after oral administration of three dosages. The analyte was separated on an Acquity UPLC BEH C₁₈ column with a total running time of 3 min using a mobile phase of 0.1% formic acid aqueous solution and methanol (80:20, v/v) at a flow‐rate of 0.25 mL/min. The calibration curves for fuziline showed good linearity in the concentrations ranging from 1 to 200 ng/mL with correlation coefficients >0.997. The precision, accuracy, recovery and stability were deemed acceptable. The method was applied to a pharmacokinetics study of fuziline in rats. The mean half‐life was 5.93, 6.13 and 5.12 h for 1, 2 and 4 mg/kg oral administration of fuziline, respectively. The peak concentration and area under the concentration–time curve increased linearly with the doses. The sum of these results indicated that, in the range of the doses examined, the pharmacokinetics of fuziline in rat was based on first‐order kinetics. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a capillary electrophoresis method for the characterization of sulfoethyl cellulose

To characterize sulfoethyl cellulose el samples, a capillary electrophoresis method was developed and validated sulfoethyl cellulose el was hydrolyzed, and the resulting d ‐glucose derivatives were analyzed after reductive amination with 4‐aminobenzoic acid using 150 mM boric acid, pH 9.5, as background electrolyte at 20°C and a voltage of 28 kV. Peak identification was derived from capillary electrophoresis with mass spectrometry using 25 mM ammonia adjusted to pH 6.2 by acetic acid as electrolyte. Besides mono‐, di‐, and trisulfoethyl d ‐glucose small amounts of disaccharides could be identified resulting from incomplete hydrolysis. The linearity of the borate buffer‐based capillary electrophoresis method was evaluated using d ‐glucose in the concentration range of 3.9–97.5 μg/mL, while limits of detection and quantification derived from the signal‐to‐noise ratio of 3 and 10 were 0.4 ± 0.1 and 1.2 ± 0.3 μg/mL, respectively. Reproducibility and intermediate precision were determined using a hydrolyzed sulfoethyl cellulose el sample and ranged between 0.2 and 8.8% for migration times and between 0.3 and 10.4% for peak area. The method was applied to the analysis of the degree of substitution of synthetic sulfoethyl cellulose el samples obtained by variation of the synthetic process and compared to data obtained by elemental analysis.     

Development and validation of a sensitive quantification method for hematoporphyrin monomethyl ether in plasma using high-performance liquid chromatography with fluorescence detection

A rapid, sensitive, precise and specific method for determination of hematoporphyrin monomethyl ether (HMME), a novel photodynamic therapy (PDT) drug, was developed and validated using high-performance liquid chromatography (HPLC) with fluorescence detection. HMME was isolated from the plasma by a single-step liquid-liquid extraction with ethyl acetate. The analyte and internal standard fluorescein were baseline separated on a Diamonsil C(18) analytical column (4.6 x 150 mm, 5 microm) and analyzed using a fluorescence detector with the excitation and emission wavelengths set at 395 and 613 nm, respectively. The method was linear in the concentration range 0.025-5 microg/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL. The inter- and intra-day accuracies and precisions were all within 10% and the mean recoveries of HMME and fluorescein were 95 +/- 3.7 and 90 +/- 2.3%, respectively. The analyte was stable during all sample storage, preparation and analysis periods. This method was successfully applied to a pharmacokinetic study after a single-dose intravenous administration of HMME (5 mg/kg) to beagle dogs. This method was reproducible and sensitive enough for the pharmacokinetic study of HMME. Based on the results of the pharmacokinetic study, we suggest that a rather long light-avoiding time is essential for patients under HMME therapy.     

Development and validation of an LC‐MS/MS method for the determination of bullatine A in rat plasma: application to a pharmacokinetic study

Bullatine A is a diterpenoid alkaloid of Xue‐Shang‐Yi‐Zhi‐Hao (Aconitum brachypodum), which is widely used in traditional Chinese medicine for the treatment of rheumatism and pain. The plasma levels of bullatine A were measured by a rapid and sensitive LC‐MS/MS method. Samples were prepared using acetonitrile precipitation and the separation of bullatine A was achieved on a Capcell Pak MG‐C₁₈ column by isocratic elution using acetonitrile (phase A) and 0.1% formic acid (phase B, pH 4.0; A:B, 30:70, v/v) as the mobile phase at a flow rate of 0.5 mL/min. Detection was performed on a triple‐quadrupole tandem mass spectrometer by multiple‐reaction monitoring of the transitions at m/z 344.2 → 105.2 for bullatine A and m/z 256.2 → 167.1 for the internal standard. The linearity was found to be within the concentration range of 1.32–440 ng/mL with a lower limit of quantification of 1.32 ng/mL. Only 1.3 min was needed for an each analytical run. This method was successfully applied in the determination of the active component bullatine A in rat plasma after intramuscular administration of A. brachypodum injection. Copyright © 2015 John Wiley & Sons, Ltd.