Development of gas chromatography/mass spectrometry following headspace solid-phase microextraction for fast determination of asarones in plasma

Asarones (alpha-asarone and beta-asarone) are the active components in the traditional Chinese medicine (TCM) of Acorus tatarinowii Schott, which has been used to treat epilepsy for several thousand years. To perform the pharmacokinetics (PK) study of alpha- and beta-asarone from the TCM essential oil, a simple, rapid and sensitive method was developed for the determination of asarones from the TCM in rabbit plasma, based on headspace solid-phase microextraction (HS-SPME) followed by gas chromatography/mass spectrometry (GC/MS) with electron ionization (EI). The extraction parameters of headspace volume, fiber coating, sample temperature, extraction time, stirring rate and ion strength were systemically optimized. Furthermore, the method linearity, detection limit and precision were also investigated. It was shown that the proposed method provided a good linearity (0.02-20 microg/mL, R(2) > 0.99), low detection limit (<2.0 ng/mL) and good precision (RSD < 7.0%). Finally, HS-SPME followed by GC/MS was applied to fast determination of alpha- and beta-asarone in rabbit plasma at different time points after oral adminstration of the essential oil from A. tatarinowii. The experimental results suggest that the proposed method provides an alternative approach to the PK studies of volatile compounds in TCMs.     

Simultaneous determination of four lignan compounds in Herpetospermum caudigerum by normal-phase high-performance liquid chromatography

A normal-phase high-performance liquid chromatographic method with diode array UV detection is developed for the simultaneous quantitation of four lignan compounds in Herpetospermum caudigerum. This analysis provides a good resolution and reproducibility. Chromatography is carried out with a mobile phase of N-hexane-dichlormethane-methanol (42.5:42.5:5, v/v) at a flow rate of 1.0 mL/min. UV detection is performed at 280 nm. The calibration curve for lignans concentration is linear over the range of 2.10 to 42.0 microg/mL, 15.26 to 305.2 microg/mL, 6.15 to 123.0 microg/mL, and 6.24 to 124.8 microg/mL, respectively. The limit of quantitation and detection for compounds 1, 2, 3, and 4 is 1.31, 2.74, 2.63, and 2.17 microg/mL and 0.28, 0.25, 0.27, and 0.31 microg/mL, respectively. The validation data show that the assay is sensitive, specific, accurate, and reproducible for the simultaneous quantitation of four compounds. This rapid method is therefore appropriate to quantitate these lignans in Herpetospermum caudigerum.     

Chemiluminescence flow sensor with immobilized reagent for the determination of pyrogallol based on potassium hexacyanoferrate(III) oxidation

A novel chemiluminescence (CL) flow-through sensor for the determination of pyrogallol has been developed. The method is based on the reaction between pyrogallol and potassium hexacyanoferrate(III) in sodium hydroxide solution. Potassium hexacyanoferrate(III) involved in the CL reaction was electrostatically immobilized on anion-exchange resin packed in a column. Pyrogallol was sensed by the CL reaction between pyrogallol and potassium hexacyanoferrate(III) which was eluted from the ion-exchange column through sodium phosphate injection. The CL emission allows quantitation of pyrogallol concentration in the range 0.01-3.8 microg/mL with a detection limit (3 sigma) of 0.003 microg/mL and a sample throughput of 118 h(-1). The relative standard deviation (n=7) was 2.2% for 0.2 microg/mL of pyrogallol. The influence of foreign compounds was tested.     

A validated chromatographic method for the determination of flavonoids in Copaifera langsdorffii by HPLC

Hydroethanolic extracts of C. langsdorffii leaves have therapeutic potential. This work reports a validated chromatographic method for the quantification of polar compounds in the hydroethanolic extract of C. langsdorffii leaves. A reliable HPLC method was developed using two monolithic columns linked in series (100 x 4.6 mm - C18), with nonlinear gradient elution, and UV detection set at 257 nm. A procedure for the extraction of flavonols was also developed, which involved the use of 70% aqueous ethanol and the addition of benzophenone as the internal standard. The developed method led to a good detection response as the values for linearity were between 10.3 and 1000 microg/mL, and those for recovery between 84.2 and 111.1%. The detection limit ranged from 0.02 to 1.70 microg/mL and the quantitation limit from 0.07 to 5.1 microg/mL, with a maximum RSD of 5.24%. Five compounds, rutin, quercetin-3-O-alpha-L-rhamnopyranoside, kaempferol-3-O-alpha-L-rhamnopyranoside, quercetin and kaempferol, were quantified. This method could, therefore, be used for the quality control of hydroethanolic extracts of Copaifera leaves and their cosmetic and pharmaceutical products.     

Validation of a simple HPLC method for assay of haplamine and its metabolites in plasma suitable for pharmacokinetic application in rats

A simple HPLC method with ultraviolet detection has been developed and validated for the simultaneous determination of haplamine and its metabolites (trans/cis-3,4-dihydroxyhaplamine) in rat. A liquid-liquid extraction was used to extract the compounds from rat plasma. The analysis was performed on a C(18) Nucleosil Nautilus column. The mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85:15; v/v) (B) used in gradient mode (38-40% B for 10 min, 40-58% B for 49 min, 58-38% B for 1 min, and 38% for 5 min) pumped at 1 mL/min. The calibration curves showed good linearity with correlation coefficients greater than 0.999 for the analytes in the investigated concentration range. The lower limit of detection was 0.007, 0.008 and 0.009 microg/mL and the lower limit of quantification was 0.014, 0.017 and 0.018 microg/mL for haplamine, and trans/cis-3,4-dihydroxyhaplamine, respectively. The method was applied to a preliminary pharmacokinetic study in rats. This method proved to meet fully the standards required of experimental pharmacokinetic studies and should be used in further preclinical investigation.     

Simultaneous determination of catechins, rutin, and gallic acid in Cistus species extracts by HPLC with diode array detection

A simple high-performance liquid chromatography method using a diode array detector (DAD) is developed for the simultaneous analysis of five major catechins: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GCT), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), and the phenolic plant metabolites gallic acid (GA) and rutin (RT) in lyophilized extracts of Cistus species. The optimal analytical conditions are investigated to obtain the best resolution and the highest UV sensitivity for the quantitative detection of catechins. The optimized conditions (acetonitrile-phosphate buffer 50mM, pH 2.5, gradient elution system on a C(18) reversed-phase column with a flow rate of 1 mL/min and UV absorbance at 210 nm) allowed a specific and repeatable separation of the studied analytes to be achieved. All compounds are successfully separated within 32 min. Calibration curves are linear in the 2-50 microg/mL range for GCT, C, and EGCG and in the 5-50 microg/mL range for GA, EGC, EC, and RT. The limit of detection values ranged from 0.24 to 0.74 microg/mL. The limit of quantitation limit values ranged from 0.77 to 1.94 microg/mL. The validated method is applied to the determination of the specific phytochemical markers GA, GCT, C, and RT in Cistus incanus and Cistus monspeliensis lyophilised extracts. The recovery values ranged between 78.7% and 98.2%. The described HPLC method appears suitable for the differentiation and determination of the most common catechins together with the glycoside rutin and the phenolic compound gallic acid and can be considered an effective and alternative procedure for the analyses of this important class of natural compounds.     

Development of HPTLC-UV absorption densitometry method for the analysis of alprazolam and sertraline in combination and its application in the evaluation of marketed preparations

A new simple, sensitive, and reproducible high-performance thin-layer chromatography method for the estimation of alprazolam and sertraline in combination is developed using silica gel plates with fluorescent indicators. The system is equipped with an automated sample applicator, and the detection was performed at 254 nm by using UV absorption densitometry. The mobile phase consists of carbon tetrachloride, methanol, acetone, and ammonia in the ratio 12:3:5:0.1. The retention factor values for alprazolam and sertraline are found to be 0.52 and 0.70, respectively. The limit of detection of alprazolam and sertraline in the mixture of given proportion is observed to be 0.05 microg/mL and 2.5 microg/mL and the limit of quantitation is 0.2 microg/mL and 10 microg/mL, respectively. The method has shown good linearity in the range of 0.2 microg/mL to 0.65 pg/mL for alprazolam (R2 > 0.9953) and 10 pg/mL to 32.5 microg/mL for sertraline (R2 > 0.9942). The intra- and inter-assay (n=5) variations in the linear range are less than 4% for alprazolam and 6% for sertraline. Three pharmaceutical products containing this combination are analyzed to test the applicability of the new method. The percentage of alprazolam and sertraline in the tablets studied range from 97.7% to 102.82% and 96.5% to 99.9%, respectively.     

Simultaneous determination of sulfamethoxazole and trimethoprim in human plasma by capillary zone electrophoresis

A capillary electrophoretic method for the simultaneous determination of sulfamethoxazole and trimethoprim in plasma was developed. Sulfamethoxazole and trimethoprim extracted from human plasma with ethyl acetate were analyzed at 20 kV and 25 degrees C using 15 mm phosphate buffer (pH 6.2) as the electrolyte. The detection was by UV at 220 nm. The run time was 8.0 min and the limit of quantification was 10.00 microg/mL for sulfamethoxazole and 2.00 microg/mL for trimethoprim. The recovery was >99% for both compounds. This method enabled the detection of sulfamethoxazole and trimethoprim in plasma of patients after oral ingestion of their combined formulation. The present simple and rapid method is applicable to drug monitoring in immunocompromised patients who are taking the combined formulation of these compounds for the treatment or prophylaxis of Pneumocystis carinii pneumonia.     

Fluorescence determination of DNA with 1-pyrenebutyric acid nanoparticles coated with beta-cyclodextrin as a fluorescence probe

A novel ultrasonication method has been successfully developed for the preparation of 1-pyrenebutyric acid (PBAC)/beta-cyclodextrin(beta-CD) complex nanoparticles. The as-prepared nanoparticles are characterized by transmission electron microscopy (TEM), fluorescence excitation and emission spectroscopy. Complex nanoparticles prepared with ultrasonication are smaller and better dispersed than single PBAC nanoparticles. At pH 3.0, the relative fluorescence intensity of complex nanoparticles of PBAC/beta-CD can be quenched by the concentration of DNA. Based on this, a novel fluorimetric method has been developed for rapid determination of DNA. In comparison with single organic fluorophores, these nanoparticle probes are better water-solubility, more stable and do not suffer from blinking. Under optimum conditions, the calibration graphs are linear over the range 0.2-15 microg mL(-1) for calf thymus DNA (ct-DNA) and 0.3-12 microg mL(-1) for fish sperm DNA (fs-DNA). The corresponding detection limit is 0.01 microg mL(-1) for ct-DNA and 0.02 microg mL(-1) for fs-DNA. The relative standard deviation of seven replicate measurements is 1.2% for 2.0 microg mL(-1) ct-DNA and 1.4% for 2.0 microg mL(-1) fs-DNA, respectively. The method is simple and sensitive. The recovery and relative standard deviation are very satisfactory. A mechanism proposed to explain the process also has been studied.     

Determination of ephedrine and related compounds in pharmaceutical preparations by ion chromatography with direct conductivity detection

An ion chromatographic method with conductivity detection for the simultaneous determination of ephedrine, pseudoephedrine and norephedrine was developed. A mixture of 2.0 mmol/L HNO3 and 2% (v/v) acetonitrile was used as eluent. The three ephedrine-like compounds were separated and determined within 20 min. The linear ranges were 0.08-50 microg/mL for ephedrine, 0.08-40 microg/mL for pseudoephedrine and 0.06-40 microg/mL for norephedrine. The detection limits were 0.03 microg/mL for ephedrine and pseudoephedrine, and 0.02 microg/mL for norephedrine. The method has been applied successfully to the determination of these sympathomimetics in pharmaceutical preparations and in Ephedra herbs.     

Simultaneous quantification of six ephedrines in a Mahwang preparation and in urine by high-performance liquid chromatography

A rapid and reliable high-performance liquid chromatographic method was developed and validated for the simultaneous determination of norephedrine (NE), norpseudoephedrine (NPE), ephedrine (E), pseudoephedrine (PE), methylephedrine (ME) and methylpseudoephedrine (MPE) in both a Mahwang traditional Chinese medicine (TCM) preparation and in urine using alpha-ethylbenzylamine as the internal standard. The method uses a Spherisorb C(18) column for an isocratic elution in a tetraethylammoniumphosphate-methanol mobile phase at a wavelength of 206 nm. The limits of detection of NE, NPE, E, PE, ME and MPE in sample solutions ranged from 0.1 to 0.3 microg[sol ]mL at a signal-to-noise ratio of 3. The within-day precision as calculated from the Mahwang TCM preparation and urine samples was below 6.2 and 1.4% for each analyte. The between-day precision as calculated from the Mahwang TCM preparation and urine samples was below 6.8 and 5.9% for each analyte. The between-day accuracy as determined from the Mahwang TCM preparation and urine samples was below 2.2 and 6.8% for each analyte. The recoveries for six compounds, obtained with compounds spiked into the Mahwang TCM preparation and urine, were found to be more than 93.6%. This method can be successfully applied to doping and excretion rate studies.     

Determination of atractylenolide II in rat plasma by reversed-phase high-performance liquid chromatography

A method for quantitative determination of atractylenolide II in rat plasma using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with UV spectrometry was established. From a variety of compounds and solvents tested, atractylenolide III was selected as the internal standard (IS) and ethyl acetate was found to be the best solvent for extracting atractylenolide II from plasma samples. RP-HPLC analysis of the extracts was performed on an analytical column (DIKMA ODS, 150 x 4.6 mm; i.d., 5 microm) equipped with a security guard pre-column system. There was good linearity over the range 0.05-5.0 microg/mL (r > 0.99). The recoveries were more than 90.0% in plasma, and the intra- and inter-day coefficients of variation were less than 10.0% in all cases. The limit of detection (LOD) was 0.025 microg/mL and the lower limit of quantification (LLOQ) was 0.05 microg/mL. The RP-HPLC method was applied to quantitate atractylenolide II in rat plasma within 24 h in a pharmacokinetics study where experimental rats received a single dose of atractylenolide II (60 mg/kg).     

High-performance liquid chromatography of lactose with evaporative light scattering detection, applied to determine fine particle dose of carrier in dry powder inhalation products

A method for quantification of the fine particle dose of lactose is described, using a hydrophilic interaction chromatography (HILIC) method and evaporative light scattering detection. The HILIC method used an aminopropyl column and a mobile phase consisting of acetonitril/water (80/20, v/v) for isocratic elution. Sensitive chromatography was obtained using a low concentration of water in the extraction solvent. The detection limit (RSD<10%) at an injection volume of 10 microL is 10 microg/mL. Linearity was obtained in the range of 10-80 microg/mL (R(2)>0.99). A relative standard deviation (RSD) of 0.5% (N=6) demonstrated good precision of the optimized method.     

Simultaneous quantification of seven bioactive components in Caulis Lonicerae Japonicae by high performance liquid chromatography

This study presents a new HPLC method for the simultaneous determination of seven major components, namely chlorogenic acid, caffeic acid, loganin, sweroside, secoxyloganin, rutin and luteolin 7-O-glucoside in Caulis Lonicerae Japonicae, a commonly used traditional Chinese medicinal herb derived from the caulis of Lonicera japonica Thunb. These seven compounds, belonging to the chemical types of phenolic acids, iridoids and flavonoids, were separated on a C18 column (250 x 4.6 mm, 5.0 microm) with the column temperature at 30 degrees C. The mobile phase was composed of (A) aqueous acetic acid (0.4%, v/v) and (B) acetonitrile using a gradient elution of 10% B at 0-12 min, 10-17% B at 12-25 min and 17% B at 25-35 min. The flow rate was 1.0 mL/min and detection wavelength was set at 245 nm. The limit of detection (S/N = 3) ranged from 0.10 to 0.23 microg/mL and the limit of quantification (S/N = 10) ranged from 0.69 to 3.56 microg/mL. All calibration curves showed good linear regression (r2 > 0.9990) within the test ranges. The intra- and inter-day precisions as determined from sample solutions were below 1.24 and 2.28%, respectively. The recoveries for seven compounds were found to range from 94.2 to 103.6%. This verified method has been successfully applied to evaluation of commercial samples of Caulis Lonicerae Japonicae from different markets in China.     

Enantioselective HPLC-UV method for determination of eslicarbazepine acetate (BIA 2-093) and its metabolites in human plasma

Eslicarbazepine acetate (BIA 2-093) is a novel central nervous system drug undergoing clinical phase III trials for epilepsy and phase II trials for bipolar disorder. A simple and reliable chiral reversed-phase HPLC-UV method was developed and validated for the simultaneous determination of eslicarbazepine acetate, oxcarbazepine, S-licarbazepine and R-licarbazepine in human plasma. The analytes and internal standard were extracted from plasma by a solid-phase extraction using Waters Oasis HLB cartridges. Chromatographic separation was achieved by isocratic elution with water-methanol (88:12, v/v), at a flow rate of 0.7 mL/min, on a LichroCART 250-4 ChiraDex (beta-cyclodextrin, 5 microm) column at 30 degrees C. All compounds were detected at 225 nm. Calibration curves were linear over the range 0.4-8 microg/mL for eslicarbazepine acetate and oxcarbazepine, and 0.4-80 microg/mL for each licarbazepine enantiomer. The overall intra- and interday precision and accuracy did not exceed 15%. Mean relative recoveries varied from 94.00 to 102.23% and the limit of quantification of the assay was 0.4 microg/mL for all compounds. This method seems to be a useful tool for clinical research and therapeutic drug monitoring of eslicarbazepine acetate and its metabolites S-licarbazepine, R-licarbazepine and oxcarbazepine.     

以睾酮为探针采用高效液相色谱法测定细胞色素CYP450 3A4的酶活性

建立了一种快速、高效的以睾酮作为探针药物评价细胞色素P450 3A4(CYP3A4)酶活性的高效液相色谱-紫外检测方法。采用的色谱柱为Phenomenex C18柱(4.6 mm×150 mm,5 μm),梯度洗脱,流速1.0 mL/min,紫外检测波长245 nm,柱温30 ℃。睾酮与大鼠肝微粒体温孵后,过已活化好的C18固相萃取小柱,收集甲醇洗脱液,于37 ℃水浴中通N2吹干,用50%甲醇复溶后进样分析测定。研究结果表明,6β-羟基睾酮的 保留时间为11.60 min,线性范围为0.5～32 μg/mL,最低检出质量浓度为0.02 μg/mL,提取率为88.41%～92.73%,方法的回收率为99.07%～101.30%;睾酮的保留时间为19.27 min,线性范围为0.5～40 μg/mL,最低检出质量浓度为0.01 μg/mL,提取率为89.59%～92.66%,方法的回收率为96.50%～98.03%。两者的日内、日间相对标准偏差均小于10%,温孵体系中的其他内源性物质不干扰测定。该方法快速、稳定、灵敏度高,适合体外睾酮及其代谢物6β-羟基睾酮的测定,可应用于体外CYP3A4酶活性的评价及酶动力学的研究。     

Flow injection analysis: Rayleigh light scattering technique for total protein determination

A novel flow injection analysis (FIA) method with Rayleigh light scattering (RLS) detection was developed for the determination of total protein concentrations. This method is based on the weak intensity of RLS of bromothymol blue (BB) (3',3"-dibromothymolsulfonephthalein) which can be enhanced by the addition of protein in weakly acidic solution. A common spectrofluorimeter was used as a detector. It was proved that the application of this method to quantify the total proteins in real samples by using bovine serum albumin was possible. The RLS signal was detected at lambda(ex)= lambda(em)=572 nm. The linear range was 7.0-70.0 microg mL(-1), the detection limit was 3.75 microg mL(-1), the reproducibility was 5.5% (n=7), and the sample throughput was 26 h(-1).     

Design and evaluation of a multi-detection system composed of ultraviolet, evaporative light scattering and inductively coupled plasma mass spectrometry detection for the analysis of pharmaceuticals by liquid chromatography

Reversed-phase liquid chromatography was coupled to a multi-detection system composed of ultraviolet (UV) detection, evaporative laser scattering detection (ELSD) and inductively coupled plasma mass spectrometry (ICP-MS). By applying the principle of post-column solvent compensation, the organic modifier content was kept constant in ELSD and ICP-MS under gradient elution. Chlorine ((35)Cl), bromine ((79)Br and (81)Br) and sulfur ((34)S) were monitored in several pharmaceutical compounds. The limit of quantitation (LOQ) was 80 ng/mL for chlorine (chlorpropamide) and 2 ng/mL for bromine (bromazepam). Calibration graphs were linear from 1.0 microg/mL to 100 microg/mL for chlorpropamide (r(2) 0.990) and from 10 ng/mL to 500 ng/mL for bromazepam (r(2) 0.996). The low LOQ value for bromine allows to quantify bromine in pharmaceutical samples below the 0.05% level of the active pharmaceutical ingredient.     

A simple and rapid determination of valproic acid in human plasma using a non-porous silica column and liquid chromatography with tandem mass spectrometric detection

A liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay was developed and validated to determine valproic acid in human plasma. The method involved a solid-phase extraction of valproic acid and betamethasone valerate, an internal standard, from plasma and detection using an LC-MS/MS system with electrospray ionization source in negative ion mode. Separation was achieved within 3 min on a non-porous silica column with mobile phase containing ammonium acetate and methanol. Multiple reaction monitoring was utilized for detection monitoring at 142.89-142.89 for valproic acid and 457.21-457.21 for the internal standard. The calibration curve for valproic acid was linear over the range of 0.5-150 microg/mL. The limit of detection was 0.17 microg/mL and the lower limit of quantification was 0.5 microg/mL, when 0.2 mL plasma was used for extraction. The percentage coefficient of validation for accuracy and precision (inter- and intra-day) for this method was less than 9.5% with recovery ranging from 82.3 to 86.9% for valproic acid.     

Determination of ceftriaxone sodium in pharmaceutical formulations by flow injection analysis with acid potassium permanganate chemiluminescence detection.

Based on the chemiluminescence (CL) emission generated from the oxidation of ceftriaxone sodium alkali hydrolysate by potassium permanganate in polyphosphoric acid (PPA), a novel determination method for ceftriaxone sodium was developed by using a flow-injection technique. The calibration curve appears to be linear in the range between 0.05 and 100 microg mL(-1) with a detection limit (3sigma) of 25 ng mL(-1), and a relative standard deviation (RSD) of 0.6% for eleven replicate determinations of 5.0 microg mL(-1) ceftriaxone sodium. The proposed method has been successfully utilized for the determination of ceftriaxone sodium in pharmaceutical formulations, while the chemiluminescence reaction mechanisms were investigated.