Liquid chromatography-electrospray mass spectrometric studies of ginkgolides and bilobalide using simultaneous monitoring of proton,ammonium and sodium adducts

A liquid chromatographic-electrospray mass spectrometric method was developed for the determination of ginkgolides and bilobalide and was applied to the analysis of commercial products of Ginkgo biloba leaf extracts. Adducts of these compounds with ammonium, proton and sodium were detected and their relative abundance depended on the electrospray fragmentor voltage. The relative standard deviation (RSD) was improved from > 17% to < 6%, when three adduct ions of (M + H)+, (M + NH4)+ and (M + Na)+ were used for quantification compared with single ion monitoring. The characteristic mass spectra of bilobalide were different from those of ginkgolides; in particular, dimers of (2M + Na)+ were observed for bilobalide only. Analysis of 26 commercial ginkgo products revealed large variations in the composition and concentrations of ginkgolides and bilobalide in herbal products.     

Capillary gas chromatographic-mass spectrometric method for the identification and quantification of some benzodiazepines and their unconjugated metabolites in plasma

A gas chromatographic-mass spectrometric method for the identification and/or quantification of diazepam, clobazam, flunitrazepam, triazolam, midazolam, oxazepam and lorazepam and some of their desmethylated and hydroxylated metabolites in plasma is described. Benzodiazepines were extracted from plasma with butyl acetate at pH 9; the hydroxylated compounds were then silylated with N,O-bis (trimethylsilyltrifluoroacetamide). Analysis was performed using a compact mass-selective detector operating in the electron-impact mode. Depending on the concentration, identification was performed either by direct comparison of the observed mass spectra with reference spectra or by the relative intensities of the most intense and characteristic ions in the selected-ion monitoring (SIM) mode. Quantification was performed in the SIM mode using the most intense ion. The intra-assay precision and accuracy were better than 5-6%; linearity was satisfactory up to 1-2 micrograms/ml. The detection limit was 1-5 ng/ml for most of the benzodiazepines. This method can be easily used in clinical situations when a safe and rapid response is essential for patient treatment.     

Development and validation of an ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry method for the simultaneous determination of selected flavonoids in Ginkgo biloba

A rapid and sensitive ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous determination of 13 flavonoids in leaf, stem, and fruit extracts of male and female trees of Ginkgo biloba to investigate gender‐ and age‐related variations of flavonoids content. Chromatographic separation was accomplished on an Acquity UPLC BEH C₁₈ column (50 mm × 2.1 mm id, 1.7 μm) in 5 min. Quantitation was performed using negative electrospray ionization mass spectrometry in multiple reaction monitoring mode. The calibration curves of all analytes showed a good linear relationship (r² ≥ 0.9977) over the concentration range of 1–1000 ng/mL. The precision evaluated by an intra‐ and interday study showed RSD ≤ 1.98% and good accuracy with overall recovery in the range from 97.90 to 101.09% (RSD ≤ 1.67%) for all analytes. The method sensitivity expressed as the limit of quantitation was typically 0.25–3.57 ng/mL. The results showed that the total content of 13 flavonoids was higher in the leaf extract of an old male Ginkgo tree compared to young female Ginkgo trees.     

Development and validation of a highly sensitive gas chromatographic-mass spectrometric screening method for the simultaneous determination of nanogram levels of fentanyl, sufentanil and alfentanil in air and surface contamination wipes

A highly sensitive gas chromatographic-mass spectrometric (GC-MS) analytical method for the determination of the opioid narcotics fentanyl, alfentanil, and sufentanil in industrial hygiene personal air samples and surface contamination wipes was developed and comprehensively validated. Sample preparation involved a single step extraction of the samples with methanol, fortified with a fixed amount of the penta-deuterated analogues of the opioid narcotics as internal standard. The GC-MS analytical procedure using selected ion monitoring (SIM) was shown to be highly selective. Linearity was shown for levels of extracted wipe and air samples corresponding to at least 0.1-2 times their surface contamination limit (SCL) and accordingly to 0.1-2 times their time weighted average occupational exposure limit (OEL-TWA) based on a full shift 9601 air sample. Extraction recoveries were determined for spiked air samples and surface wipes and were found to be quantitative for both sampling media in the entire range studied. The air sampling method's limit of detection (LOD) was determined to be 0.4 ng per sample for fentanyl and sufentanil and 1.6 ng per sample for alfentanil, corresponding to less than 1% of their individual OEL for a full shift air sample (9601). The limit of quantification (LOQ) was found to be 1.4, 1.2, and 5.0 ng per filter for fentanyl, sufentanil, and alfentanil, respectively. The wipe sampling method had LODs of 4 ng per wipe for fentanyl and sufentanil and 16 ng per wipe for alfentanil and LOQs of respectively, 14, 12, and 50 ng per wipe. The analytical intra-assay precision of the air sampling and wipe sampling method, defined as the coefficient of variation on the analytical result of six replicate spiked media was below 10 and 5%, respectively, for all opioids at all spike levels. Accuracy expressed as relative error was determined to be below 10%, except for alfentanil at the lowest spike level (-13.1%). The stability of the opioids during simulated air sampling was investigated. For fentanyl and sufentanil a quantitative recovery was observed at all spike levels, while for alfentanil recoveries ranged from 60.3 to 85.4%. When spiked air samples were stored at ambient temperature and at -15 degrees C quantitative recovery was found for fentanyl and sufentanil after 7 and 14 days. For alfentanil a slight loss seemed to occur upon storage during 7 days, being more explicit after 14 days. Ambient storage of spiked wipes seemed to lead to significant losses of all opioids studied, yielding recoveries of 37.7-88.3%. Upon storage of similar wipes at -15 degrees C a significantly higher recovery was found ranging from 77.3 to 88.3%. The developed analytical and sampling procedures have been recently applied in an explorative field study of which the results of surface contamination wipe sampling are presented in this paper. To our knowledge, this is the first study addressing the development and validation of analytical procedures for the assessment of external occupational exposure to potent opioid narcotics.     

Development of a solid-phase extraction/gas chromatographic-mass spectrometric method for quantification of succinic acid in nucleoside derivatives for oligonucleotide synthesis

A solid-phase extraction (SPE)/gas chromatographic-mass spectrometric (GC-MS) method was developed for analysing residual succinic acid in nucleoside derivatives to be used in oligonucleotide synthesis. Use of a SPE protocol, enabled most of the derivatives to be trapped, thereby creating eluates enriched in succinic acid. GC-MS was used to quantify the amount of residual succinic acid in four different nucleoside preparations, with succinate concentrations varying from 0.18 to 0.24% (w/w). The within-day repeatability of the method was found to be 1.25% RSD. A linear relationship was observed between the amount of succinic acid in the sample and the GC-MS peak area, with a correlation coefficient of 0.9997 in the concentration interval 0.05-2.5% (w/w). Recoveries were measured by the addition of internal standards to working solutions and varied between 99.8 and 102.6%.     

Development and validation of a gas chromatographic—mass spectrometric procedure for the identification and quantification of residues of chloramphenicol

A method for the identification and quantification of residues of the antibiotic chloramphenicol was developed and validated. The method is based on combined gas chromatography-mass spectrometry with negative-ion chemical ionization and the use of [³⁷Cl₂]chloramphenicol as an internal standard. A set of identification criteria, in accordance with guidelines of the European Community, is described. For urine, muscle and eggs limits of detection and quantification of 0.1 μg kg^?1 are obtained. The method shows good repeatability and reproducibility. Results for urine were compared with those obtained with a radioimmunochemical procedure and an enzyme immunoassay (Quik-Card). Screening with an immunochemical procedure followed by confirmation with gas chromatography-mass spectrometry was found to be an effective strategy for monitoring residues of chloramphenicol in biological matrices.     

A gas chromatographic-mass spectrometric method for trace analysis of chlorofluorocarbons and their replacement compounds in atmospheric samples

Summary The simultaneous determination of chlorofluorocarbons and their replacement compounds in ambient air samples requires the preconcentration of appropriate air volumes on suitable adsorbents. The performance of a dual-layer adsorption tube to be used in this type of analysis was evaluated in terms of breakthrough volumes (BTV) for the compounds of interest at ambient temperature. To verify the effectiveness of such adsorption tubes, real air samples were enriched at ambient temperature and were subsequently analyzed by capillary gas chromatography coupled with quadrupole mass spectrometry, operating in SIM mode. The results obtained confirmed the efficacy of the proposed method, in terms both of sensitivity and chromatographic separation efficiency.     

Qualitative and quantitative analysis of phenolic acid glycosides in Ginkgo biloba L. leaf,G. biloba leaf extract and its injection

Ginkgo biloba L. leaf (GBL) is one of the most commonly used medicinal plants in the world. Phenolic acids with biological activities have a relatively high content in G. biloba leaf extracts (GBE); therefore they are of great significance for the quality control of GBL, GBE and its preparations. However, there have been few studies focused on their analysis. In this work, 12 phenolic acids, including 11 phenolic acid glycosides, were identified by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC–Q-TOF/MS). Then, a method combining enzymolysis with HPLC was established for quantification of phenolic acid glycosides. It was found that the aglycones of phenolic acid glycosides mainly comprised five phenolic acids: 2,4,6-trihydroxybenzoic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid and p-coumaric acid. The quantitative method was validated, and the correlation coefficient (0.9993–0.9999), recovery (≥88.4%), repeatability (≤0.8%), and inter-day precision (≤5.5%) were satisfactory. Finally, the contents of glycosides of five phenolic acids in GBL, GBE and GBE injection from different sources were determined by the developed method. The method was accurate, repeatable and practicable, which could be helpful for the quantification of phenolic acid glycosides in other products containing GBL or GBE.     

Development and validation of an MEKC method for determination of nitrogen-containing drugs in pharmaceutical preparations

A fast and accurate micellar electrokinetic capillary chromatography method was developed for quality control of pharmaceutical preparations containing cold remedies as acetaminophen, salicylamide, caffeine, phenylephrine, pseudoephedrine, norephedrine and chlorpheniramine. The method optimization was realized on a Beckman P/ACE System MDQ instrument. The baseline separation of seven analytes was performed in an uncoated fused silica capillary internal diameter (ID)=50 microm using tris-borate (20 mM, pH=8.5) containing sodium dodecyl sulphate 30 mM BGE. On line-UV detection at 214 nm was performed and the applied voltage was 10 kV. The operating temperature was 25 degrees C. After experimental conditions optimization, the proposed method was validated. The evaluated parameters were: precision of migration time and of corrected peak area ratio, linearity range, limit of detection, limit of quantification, accuracy (recovery), ruggedness and applicability. The method was then successfully applied for the analysis of three pharmaceutical preparations containing some of the analytes listed before.     

Validated HPLC method for simultaneous estimation of khellol glucoside,khellin and visnagin in Ammi visnaga L. fruits and pharmaceutical preparations

Tea bags including fruits of Ammi visnaga L. are used in Egypt as remedy for the treatment of kidney stones. Our study focuses on developing simple and rapid method utilising HPLC for quantitative estimation of khellol glucoside (KG), khellin (KH) and visnagin (VS) simultaneously. Their concentrations were determined in A. visnaga L. fruits at different developmental stages and in pharmaceutical formulations together with following up them during shelf life. Separation was accomplished using HPLC. Perfect resolution between KG, KH and VS was possible through using a mobile phase consisting of water:methanol:tetrahydrofuran (50:45:5, v/v/v). Peaks were detected at 245 nm. The suggested method for the determination of KG, KH and VS was successful in determining the analytes of interest without any interference of other compounds and matrix. All validation parameters were satisfactory and the procedure was relatively easy and fast as extracts are evaluated without previous steps of purification.     

Direct extraction of alkylphenols, chlorophenols and bisphenol A from acid-digested sediment suspension for simultaneous gas chromatographic-mass spectrometric analysis

The direct extraction of alkylphenols, chlorophenols and bisphenol A from an acid-digested sediment suspension for GC-MS analysis was studied. The sediment was digested with acid while the hydrolyzed analytes were being extracted with dichloromethane. The conditions of the acid digestion and extraction were optimized in terms of time, acidity of digestion, and extracting solvent. It is possible to complete the extraction within 20 min with 5 ml of 0.1 M HCl digesting solution and three portions of 5 ml of dichloromethane. The recoveries of analytes were mostly around 90% with about 10% relative standard deviations. With this technique parallel treatment of large numbers of sediment samples is possible without any expensive special equipment or heating process. The analytical characteristics of this extraction technique were compared with Soxhlet extraction and the pressurized liquid extraction technique. The technique was examined and evaluated for real environmental sediment samples and certified reference material of natural matrix.     

Development and validation of an UHPLC-LTQ-Orbitrap MS method for non-anthocyanin flavonoids quantification in Euterpe oleracea juice

Euterpe oleracea fruits have gained much attention because of their phenolic constituents that have shown potential health benefits. The aim of this work was to quantify the major non-anthocyanin flavonoids (NAF) in the fruit juice by an accurate method coupling ultra-high pressure liquid chromatography with a linear ion trap-high resolution Orbitrap mass spectrometry system (UHPLC-LTQ-Orbitrap MS). Fruits were processed to juice, and then the juice was lyophilized and defatted. The residue was then extracted in the presence of methanol by sonication. The extraction time was optimized and recovery rates of the extraction were >90 %. The extracts were dried and solubilized again in 40 % MeOH, which showed the best compromise for MS detection. For the UHPLC quantification, a HSS C18 column (1.8 μm) was used with a gradient elution of methanol and water both with 0.1 % formic acid. Total error and accuracy profiles were used as validation criteria. Seven compounds and their isomers were successfully separated, including the major NAF. Calibration in the matrix was found to be more accurate than calibration without matrix. Trueness (<15 % relative bias), repeatability, and intermediate precision (<13 % RSD), selectivity, response function, linearity, LOD (ranged from 0.04 to 0.81 μg/mL) and LOQ (0.15–5.78 μg/mL) for 12 compounds were evaluated and the quantification method was validated. Its applicability was demonstrated on real samples from different suppliers. Their qualitative and quantitative profiles were similar and some compounds were for the first time quantified. In addition, eriodictyol was identified for the first time in this fruit along with five other flavonoids for which possible structures were proposed.     

Development, optimization and validation of a fingerprint of Ginkgo biloba extracts by high-performance liquid chromatography

A four-step development, optimization and validation strategy for high-performance liquid chromatography (HPLC) fingerprints of Ginkgo biloba extract is described. A suitable chromatographic system was selected first. The following step was performing a screening design to select important parameters. After selecting some controllable parameters and their range to further optimize, gradient optimization with uniform design was done. At last, method validation including determination of injection precision, repeatability, and a sample stability test, was performed. Through this effective and integrated four-step method, a feasible and reliable HPLC fingerprint to identify and assess the Ginkgo biloba quality can easily be established using a linear gradient elution with acetonitrile/0.1% phosphoric acid (from 14/86 to 30/70, v/v, in 40 min) as mobile phase, a column temperature of 30 degrees C and a detection wavelength of 350 nm. The strategy can also be applied for the development of fingerprints in the quality control of other herbal medicines.     

Development and validation of an HPTLC method for simultaneous quantitation of isoorientin, isovitexin, orientin, and vitexin in bamboo-leaf flavonoids

A simple HPTLC method has been developed for the simultaneous determination of isoorientin, isovitexin, orientin, and vitexin, both pure and in commercial samples of bamboo-leaf flavonoids. The flavone C-glycosides, including isoorientin, isovitexin, orientin, and vitexin, were extracted from bamboo-leaf flavonoids with methanol and chromatographed on silica gel 60 plates in an automatic developing chamber with tetrahydrofuran-toluene-formic acid-water (16 + 8 + 2 + 1, v/v/v/v) mobile phase. Quantitation was obtained with UV detection at 350 nm. Polynomial calibration plots were constructed in the concentration range 200-1200 ng/zone for isoorientin, 100-600 ng/zone for isovitexin, 160-960 ng/zone for orientin, and 30-360 ng/zone for vitexin with good correlation coefficients (r > or = 0.9995). The method was validated for precision (interday and intraday), repeatability, and accuracy. Accuracy of the method was evaluated by a recovery study conducted at three different levels, and the average recovery was found to be 93.95% for isoorientin, 95.30% for isovitexin, 99.79% for orientin, and 100.46% for vitexin. The proposed HPTLC method for estimation of isoorientin, isovitexin, orientin, and vitexin was found to be simple, precise, specific, and accurate and can be used for manufacturing QC of bamboo-leaf flavonoids or for governmental regulatory purposes.     

Qualitative and quantitative assessment of the HPLC fingerprints of Ginkgo biloba extract by the involution similarity method.

In this paper, we develop a new method for evaluating chromatographic fingerprints of traditional Chinese medicine (TCM) by the average involution similarity and the quantitative involution similarity. To validate this novel approach, we studied the chromatographic fingerprints of Ginkgo biloba extract by the new similarity parameters. The results were compared with those of the cosine of vectorial angle (S(F)), the correlative coefficient (r) and some other quantitative parameters, such as the apparent quantitative similarity of fingerprint R% and the average mass percentage M%. The approach represented in this paper was proved to well reflect the similarity alteration of each batch of Ginkgo biloba extract and the quantitative differences of the extracted constituents. The S(gxq) and S(gxsq) are the best qualitive and quantitative similarity parameters of all those mentioned in this paper; they can be profitably used for the qualitative and quantitative assessments of TCM chromatographic fingerprints. Through this study, the quality evaluation of TCM can be simplified by using only one parameter of with the qualitive and quantitative functions proposed in this paper.     

Development and validation of a high-performance liquid chromatography-tandem mass spectrometric method for quantification of daunorubicin in rat plasma

A high-performance liquid chromatography-tandem mass spectrometric method (LC-MS/MS) has been developed and validated for the determination of daunorubicin in K₃EDTA rat plasma. The 100 μL plasma samples were extracted by a methanol:acetone protein precipitation step in the presence of additional 50 μL of 70% (w/v) zinc sulfate, and subsequently analyzed by LC/MS/MS using positive turbo-ion spray ionization mode. The LC/MS/MS instrument was operated in the multiple-reaction-monitoring (MRM) mode. Doxorubicinol was better than doxorubicin as the internal standard because its recovery and absolute matrix effect data exactly matched with those for daunorubicin. In addition, HPLC gradient condition was optimized to thoroughly separate daunorubicin from the background interference. The validated concentration range was from 0.250 to 100 ng/mL. The true recoveries of daunorubicin and doxorubicinol were 93.2% and 93.6%, respectively. In addition, the ion-suppression data of daunorubicin and doxorubicinol were 78.2% and 78.4%, respectively. Absence of the relative matrix effect from six unique lots was confirmed. Results obtained from the GLP validation study demonstrated very good accuracy (95-105%) and precision (less than 10% CV).     

Development and validation of a RP-HPLC method for simultaneous quantification of bedaquiline (TMC207), moxifloxacin and pyrazinamide in a pharmaceutical powder formulation for inhalation

A reversed phase high performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of bedaquiline (TMC207), moxifloxacin and pyrazinamide in a pharmaceutical powder formulation for inhalation has been developed and validated. The powder was simply dissolved in methanol and the analytes separated in a run time of 20?min on a Luna C18 (2) (150?×?4.6?mm, 5?µm) column using gradient elution with methanol and triethylamine phosphate buffer (pH 2.5) delivered at 1.2?mL/min. The detection (with retention time) was carried out at 269?nm (2.9?min) for pyrazinamide, 296?nm (7.0?min) for moxifloxacin and 225?nm (16.3?min) for bedaquiline, respectively. The method was linear for all analytes in the concentration range 1-100?µg/mL with correlation coefficients >0.998. Lower limits of quantitation (µg/mL) of bedaquiline, moxifloxacin and pyrazinamide were 0.56, 0.43 and 0.24, respectively. The method was accurate (relative error in the range ?0.2 to 2.2) and precise (%RSD ≤6.2) with recovery in the range 100.0–104.7%. The method was successfully applied to determine the drug content and content uniformity of the three analytes in a spray-dried combination powder formulation for inhalation containing L-leucine.     

Liquid chromatographic-mass spectrometric analysis for screening of patients with cystinuria, and identification of cystine stone.

Analyses of amino acids in the urine of a normal human and of patients with heterozygous and homozygous cystinuria have been carried out, using liquid chromatography-mass spectrometry with an atmospheric pressure ionization interface system. A kidney cystine stone was also analysed by this system. Very intense quasi-molecular ions ([M + H]+) of standard cystine, arginine, lysine and ornithine were observed on mass chromatograms as base peaks. Mass chromatograms of the urine samples from a normal human and from patients with heterozygous and homozygous cystinuria were easily distinguishable. The retention times in the mass chromatogram and mass spectrum of kidney stone cystine was almost the same as that of authentic cystine.     

Development,method validation and simultaneous quantification of eleven bioactive natural products from high-altitude medicinal plant by high-performance liquid chromatography

Herein, a novel, rapid, reliable, simple method validation and simultaneous quantification of 11 bioactive compounds (mostly xanthones) have been described. International Conference on Harmonization guidelines were used for the analytical method validation. Good linearity, repeatability, intra- and inter-day precision, accuracy and reliability were well-illuminated in the method validation procedure. The calibration curves showed a good linear relationship (r > 0.999) within test range. Precision was evaluated by intra- and inter-day tests with relative standard deviation <2.79% and accuracy validation recovery of 74.16%–91.84%. On quantification study, the validated method described the high content of bioactive xanthone derivatives, including 1-hydroxy-3, 5-dimethoxyxanthone ( 7 ), 2-(allyloxy)-8-hydroxy-1, 6-dimethoxyxanthone ( 6 ) 1, 7, 8-trihydroxy-3-methoxyxanthone ( 9 ) and Coxanthone E ( 5 ) in Codonopsis ovata, which is advantageous given the numerous pharmacological and biological effects associated with these compounds, which mostly exhibit anti-cancerous, antioxidant, anti-inflammatory, anti-mutagenic and anti-obesity effects. The bulk abundance of these compounds can also be used for further modification to produce better lead molecules for drug discovery with low toxicity and high potency. The proposed method makes it possible to simultaneously determine all bioactive compounds in one run and can be extended to marker-based standardization of herbal formulations in medicinal and pharmaceutical industries.     

Development and validation of a new Fourier transform infrared spectrometric method for the quantification of urea in creams and ointments

A new Fourier transform infrared (FTIR) spectrometric method for the quantification of urea in creams and ointments was developed and validated. The sample was solubilized in a mixture of chloroform/acetonitrile (1/1 v/v) which allows to solubilize the vaseline or other fats as well as urea and water. The solution was examined in a transmission cell with a pathlength of 500 μm provided with NaCl windows. The urea spectrum was obtained by subtraction of the reference solvent mixture spectrum and the reference water spectrum from the sample spectrum. The absorbance of the peak at ±1688 cm⁻¹ was compared to those of calibration standards to quantify urea. The detection limit and the quantification limit (three times and 10 times the noise, respectively) were estimated as 0.4 and 1.3 μg ml⁻¹, respectively. Validation of the method was realized and application of this method to stability tests was done.