μL Of Mobile Phase Research Articles

Novel design of low volume mobile phase paper chromatography

A novel design and set up of paper chromatography in the form of radial serpentine arms laying horizontally is proposed. The consumption of mobile phase is reduced to be 1 mL or less as compared to the tens of mL typically used in the conventional set ups for ascending paper chromatography, without any change in the order of the separated components. Greenness measurement based on the ChlorTox value is approximately 25, when comparing to a conventional system that consumes 25 mL of mobile phase. A digital image of the chemical separation makes data analysis more feasible as the image can be kept, and hence, extending the time for data analysis. The Retention Factor (Rf) value from a serpentine shaped stationary phase can be calculated based on measurement of linear distances of the solvent front and those travelled by each component, and converting them to arclengths using the mathematical equation of a catenary wave that produces that particular design. Proof of concept is demonstrated by the separation of food dyes using a two solvent system, where a nonvolatile salt solution is followed by a volatile organic solvent mixture of alcohols and base. Relationships between concentration, color intensity (RGB values), and zone arclength are also demonstrated which indicate possibility of quantitative study on the serpentine design using digital image analysis.

Enantiomeric Separation of Colchicine and Lacosamide by Nano-LC. Quantitative Analysis in Pharmaceutical Formulations

A chiral analytical methodology was developed by nano-liquid chromatography (nano-LC) enabling the enantiomeric separation of two chiral drugs, lacosamide (novel antiepileptic drug) and colchicine (antiuremic drug), commercialized as pure enantiomers. A capillary column lab-packed with an amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase was used in a lab-assembled nano-LC system. Lacosamide and colchicine enantiomers were separated in less than 8.0 and 9.0 min, respectively, with resolution values of 1.6 and 2.3, using 20 nL of sample and 1.8 µL of mobile phase per analysis. The analytical characteristics of the proposed methodology were evaluated according to the International Council for Harmonisation (ICH) guidelines, showing good analytical performance with good recoveries (97–98% and 100–103%) and precision values (relative standard deviation (RSD) <10.5 and <3.0%) for lacosamide and colchicine enantiomers, respectively. LODs were 1.7 and 2.0 µg/mL for (S)- and (R)-lacosamide, respectively, and 1.0 µg/mL for both colchicine enantiomers. Additionally, the developed methodology enabled to detect a 0.1% of the enantiomeric impurities, fulfilling the ICH regulation requirements. The method was applied to the determination of lacosamide and colchicine enantiomers in different pharmaceutical formulations to ensure their quality control. The content of the enantiomeric impurities was below a 0.1% and the amount of (R)-lacosamide and (S)-colchicine agreed with their labeled contents.

A Comprehensive HPLC-MS-MS Screening Method for 77 New Psychoactive Substances, 24 Classic Drugs and 18 Related Metabolites in Blood, Urine and Oral Fluid.

To date, more than 800 molecules are classified as New Psychoactive Substances (NPS), and it is reported that this number increases every year. Whereas several cases of polydrug consumption that led to acute intoxication and death are reported, a lack of effective analytical screening method to detect NPS and classical drug of abuse in human matrices affects the prompt identification of the probable cause of intoxication in emergency department of hospitals. In this concern, a fast, simple and comprehensive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) screening method to detect and quantify 77 NPS, 24 classic drugs and 18 related metabolites has been successfully developed and validated in blood, urine and oral fluid. A small volume (100 µL) of whole blood samples spiked with internal standard deuterated mixture was added to 70 µL of M3® buffer, and after precipitation of blood proteins, the supernatant was evaporated to dryness and reconstituted in 1 mL of mobile phase. Same volume (100 µL) of urine and oral fluid samples spiked with internal standard deuterated mix were only diluted with 500 µL of M3® reagent. One microliter of samples of each matrix was injected into HPLC-MS-MS equipment. The run time lasted 10 min with a gradient mobile phase. Mass spectrometric analysis was performed in positive ion multiple reaction monitoring mode. The method was linear for all analytes under investigation with a determination coefficient always better than 0.99. The calibration range for blood and oral fluid was from limits of quantification (LOQs) to 200 ng/mL, whereas that for urine was LOQs to 1000 ng/mL. Recovery and matrix effect were always higher than 80%, whereas intra-assay and inter-assay precision were always better than 19% and accuracy was always within 19% of target in every matrix. Applicability of the method was verified by analysis of samples from real cases.

Kavalactones and Flavokavins Profiles Contribute to Quality Assessment of Kava (Piper methysticum G. Forst.), the Traditional Beverage of the Pacific

Kava (Piper methysticum) is increasingly traded internationally and there is need for a rapid method to analyze kava raw material before export. The objectives of the present study were: (i) to develop a simple and robust protocol for high throughput simultaneous quantification of kavalactones (KLs) and flavokavins (FKs) in kava and (ii) to assess its potential for quality control. Methysticin; dihydromethysticin; kavain; desmethoxyyangonin; dihydrokavain; yangonin; and flavokavin A, B and C were quantified using HPTLC in acetonic extracts of 174 kava varieties. UHPLC analysis was conducted on a subset of six varieties representing the genetic variation of the species. The genetically distinct groups of nobles, two-day and wichmannii varieties were clearly differentiated and multivariate analyses of UHPLC and HPTLC data were congruent. Noble varieties have significantly low FKs/KLs (0.13) and high kavain/flavokavin B (K/FKB = 7.31). Two-day and wichmannii varieties are characterized by high FKs/KLs (0.36, 0.21) and low K/FKB (1.5, 1.7). A high-throughput HPTLC protocol was developed with a total analytical time of 50 min for 20 samples and only 10 mL of mobile phase. The use of acetone, sonication and two different detection wavelengths improves the accuracy compared to previous HPLC studies and confirms that kava varieties exhibit distinct chemotypes clearly differentiated by their FKs/KLs profiles. These results will strengthen the use of Codex Alimentarius regional standards.

Acetaminophen Analysis in Different Commercial Formulation of Iranian Acetaminophen Tablets

Acetaminophen is one of the most important antipyretic and analgesic drugs. It has an excellent efficacy when it's used in therapeutic doses, but in unsafe doses it can be resulted in hepatotoxicity and permanent liver failure. Due to disparities that have been reported between the actual and stated concentration of acetaminophen tablets, we aimed to compare the actual quantity of 8 Iranian generic acetaminophen tablets with the stated amount on the label, using HPLC method. Drug concentration has measured by HPLC. We performed USP procedure for all models such as tablets, capsule, and oral solution assay preparations USP-36 NF31 by standard preparations. Method validation was achieved. For USP guidelines performance we need to concern on standard references. By chromatography equipment, we used HPLC analyzer 1200 degasser, 1200 bin pump, 1200 ALS, and 1200 VWD. Acetaminophen sample solution included 325 mg weighted quantity of the powder that transferred to a 200 ml volumetric flask in addition to 100 ml of mobile phase. A part of this solution was transferred throughout a 0.5 micrometer permeable filter (or finer). By injecting 10 µL of standard solution into the chromatograph, major peaks are measured as a response. As a final point, calculation the quantity of acetaminophen in each brand was obtained via specific formula. According to the USP, all brands consisted of 90.0– 110.0 % of the labeled amount of active ingredient (p<0.05). Consequently, The USP standards are met for drugs with different lot numbers by a variety of companies. The differences of clinical attributes of Paracetamol overdose between Iran and other countries may be related to pharmacokinetic and pharmacodynamics issues, metabolism, genetic factors or environmental effects. Further studies are recommended.

DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR QUANTITATIVE ESTIMATION OF PALIPERIDONE

A planar chromatography (HPTLC) method has been developed and validated for quantitative estimation of the atypical antipsychotic drug paliperidone, which is weakly basic and poorly water soluble. The Camag HPTLC system, operated by software winCATS (ver.1.4.1.8), was used. Sample application was facilitated by Linomat 5 applicator. After sample application, plates were subjected to ascending development in twin trough chamber of size 10 cm x10 cm, using about 10 mL of mobile phase. The optimized mobile phase was composed of ethyl acetate:chloroform:toulene:methanol (2.5:2.5:2.5:2.5 v/v/v/v). In post development, the plates were air dried and then scanned densitometrically using a UV detector at 278 nm in absorbance mode. In HPTLC densitogram, well defined peak was obtained for paliperidone with starting position at 0.27 Rf, max position at 0.31 Rf and end position at 0.34 Rf. The optimal Rf value for paliperidone was found to be 0.31. Performance characteristics of proposed and newly developed HPTLC method were statistically validated as per recommendations of ICH guidelines of analytical method validation. The HPTLC method was found to be linear across the range from 20 to 140 ng/band. The LOD and LOQ values were found to be 1.288 and 3.905ng/band, respectively. The method was found to be accurate, precise, robust and economical for the determination of paliperidone from bulk and its extended release tablet formulation.

Novel strategy for fluorescence determination of glibenclamide in samples with high concentration of caffeine based on a low-pressure flow injection chromatography system

In this work, a new low-pressure flow injection chromatography (FIA-C) system with fluorescence detection was developed for the toxicological control of glibenclamide in beverages with high caffeine content. As caffeine quenched the fluorescence signal of glibenclamide, a separation of the analyte from the sample matrix was proposed as a simple and rapid strategy. The separation was performed in a commercially available monolithic column (RP-18e, 25 mm × 4.6 mm i.d.) inserted in a flow injection system. The mobile phase used for analysis was acetonitrile/acetic acid (50:50 v/v), with a flow rate of 1.03 mL min−1. For each analysis, only 5.2 mL of mobile phase was used.After the optimization of the variables of the system, a calibration curve with a linear range between 0.50 and 10.0 mg L−1 was obtained (R2 = 0.997). The precision of the proposed method was evaluated in terms of the relative standard deviation obtaining 0.58 and 1.68% for the intra-day precision and inter-day precision, respectively. The detection limit was 0.10 mg L−1 and the sample throughput, taking into account the whole procedure, was 12 h−1. The method was applied to fortified real samples with satisfactory recovery values (90.4–103.7%). On the other hand, samples adulterated with commercially GLB pills were also analyzed with very good results (96.5–104.5%).

Development of Micellar HPLC-UV Method for Determination of Pharmaceuticals in Water Samples.

Method for extraction and determination of amoxicillin, caffeine, ciprofloxacin, norfloxacin, tetracycline, diclofenac, ibuprofen, nimesulide, levonorgestrel, and 17α-ethynylestradiol exploiting micellar liquid chromatography with PDA detector and solid-phase extraction was proposed. The usage of toxic solvents was low; the chromatographic separation of the medicaments was performed using a C18 column and mobile phases A and B containing 15.0% (v/v) ethanol, 3.0% (m/v) sodium dodecyl sulfate (SDS), and 0.02 mol·L−1 phosphate at pHs 7.0 and 8.0, respectively. The method is simple, selective, and fast, and the analytes were separated in 23.0 min. For extraction, 1000 mL of sample containing 2.0% (v/v) ethanol and 0.002 mol·L−1 citric acid at pH 2.50 was loaded through a 1000 mg of C18 cartridge. The analytes were eluted using 3.0 mL of ethanol, which were evaporated and redissolved in 0.5 mL of mobile phase. Concentration factors better than 1200, except amoxicillin (224), were obtained. The analytical curves were linear (R 2 better than 0.992); LOD and LOQ (n=10) presented values in the range of 0.019–0.247 and 0.058–0.752 mg·L−1, respectively. Recoveries of 99% were obtained, and the results are in agreement with those obtained by the comparative methods.

SIMULTANEOUS ESTIMATION OF THIOCOLCHICOSIDE AND ACECLOFENAC BY HPTLC

Objective: Therefore the aim of the present work was to develop simple, precise and accurate HPTLC method for simultaneous determination of THIO and ACE in pharmaceutical dosage form and application of the method for dissolution study with help of HPTLC method.Methods: The TLC procedure was optimized in view to develop a simultaneous assay method for THIO and ACE. HPTLC Pre-coated plates silica gel 60 F254 20×10 cm, layer thickness 0.2 mm (Merck, Germany). The samples were spotted in the form of bands (8 mm) with a Camag 100 microliter sample (Hamilton) syringe on silica gel precoated aluminium 60F254 plates. The mobile phase consisted of methanol: chloroform: water (9.6: 0.2: 0.2 v/v/v) and 10 ml of mobile phase were used per chromatography run. Plates were scanned over the range of 200-400 nm and the spectra were overlain.Results: The detector response was found to be linear in the concentration range of 0.03-0.180 µg/band and 0.75-4.5 µg/band for THIO and ACE and noting the peak areas. Accuracy of the assay method was evaluated with the recovery of the standards from excipients. The mean percentage recoveries obtained for THIO and ACE were 99.34% and 99.08%, respectively, reported. The peak purity of both drugs was assessed by comparing the respective spectra of standard drugs and samples at peak start, peak apex and peak end positions of the spot.Conclusion: HPTLC, with its advantage of low operating cost, high sample thought and minimum sample preparation need is now days preferred as routine analytical techniques for control and assurance. The validated HPTLC method employed here proved to be simple, fast, accurate, precise and sensitive, thus can be used for routine analysis of Thiocolchicoside and Aceclofenac in tablet dosage form.

Abstract 1646: Synthesis of [11C]onapristone for clinical investigation

Abstract Background: Onapristone is a type I anti-progestin, which prevents the progesterone receptor (PR) monomers from dimerizing, inhibits ligand-induced phosphorylation and prevents association of the PR with its co-activators, thus preventing PR-induced transcription. Onapristone has the potential to treat patients with endometrial cancer, breast cancer, uterine sarcomas, a potential subset of ovarian cancer, prostate cancer and other tumors in which the progesterone receptor plays a role in growth, proliferation and metastasis. [11C]-radiolabeled onapristone and its visualization via PET-scan, coupled with pharmacokinetic (PK) studies, has the potential to determine tissue-specific and blood PK parameters including tumor/tissue and plasma concentrations, whole body distribution and half-life of onapristone. The aim of this study is the development of a rapid Good Manufacturing Product (GMP) synthesis of parentally administered [11C]onapristone. Methods: The production of [11C]-labeled radiopharmaceuticals used a Tracerlab® FX c-Pro (GEMS) synthesis module. Carbon-11 was produced at Institut Curie-Hôpital René Huguenin via the 14N(p,α)11C nuclear reaction using a PETTrace cyclotron (GEMS) equipped with a carbon-11 target. Carbon-11 is delivered from the cyclotron as [11C]CO2 in the synthesis module, and reduced to [11C]CH4. Methane is halogenated to [11C]CH3I and converted to [11C]methyl triflate (CH3OTf). To prepare [11C]onapristone, the module was loaded with 1mg of N-desmethyl-onapristone (Arno Therapeutics) and 500µL of DMSO in the reaction vessel (Sigma®). [11C]CH3OTf was bubbled into the reaction vessel and heated at +50°C for 10 minutes. The reaction mixture was then diluted with 1 ml of mobile phase and purified using a semi-preparative HPLC column: Sunfire C18 5µm 250X10mm (Waters®), mobile phase: acetonitrile (Sigma®)/water (Waters®) 50/50 v/v; flow rate 4 mL/min. The product fraction was collected in 40mL of sterile water and was passed through a C18 SEP PAK (Waters®). This fraction was eluted with isotonic saline (Braun®) and ethanol (Sigma®). The resulting formulation was passed through a 0.22µm sterilizing filter into a sterile dose vial. Results: 10 tests were performed. Time of synthesis was 50 minutes. The amount of carbon-11 delivered was 12-14GBq for the irradiation parameters as follows: 10 min; 10µA. The decay-corrected yield of reducing [11C]CO2 to [11C]CH4 was &amp;gt;99%. The yields of preparing [11C]CH3I were 27-29%. Time retention of [11C]onapristone was 8.5-9.5 minutes and the non-decay-corrected radiolabelling yields were 1-2%. Conclusions: We have successfully developed a fully-automated production of [11C]onapristone ready for use in clinical trials. Citation Format: Olivier Madar, Julien Fouque, Stefan Proniuk, Keyvan Rezai, Samuel Huguet, Alexander Zukiwski, Erard M. Gilles, Alice S. Bexon, François Lokiec. Synthesis of [11C]onapristone for clinical investigation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1646. doi:10.1158/1538-7445.AM2014-1646

O5: Amitriptyline poisoning of a baby: How informative can be hair analysis?

Introduction Following the hospitalization of a 6-month old baby for CNS disorders then coma, an intoxication with amitriptyline (AMI) was highlighted (blood concentrations : AMI = 99.4 μg/L and nortriptyline (NOR) = 154 μg/L). Complimentary investigations suggested a shaken baby syndrome. Given the family context, a judge ordered hair tests for both the child and his parents to document drug exposure. The aim of this case is to present the interest of hair analysis when poor evidence is available. Methods For the child, we collected a strand of hair (4 cm of length, 60 mg) during the acute intoxication (To) and a second strand 5 weeks after (5 cm of length, 82 mg). Three months after the acute intoxication of the baby, samples of hair were collected from mother (strand of 22 cm of length, 1951 mg, and black color) and from father (strand of 5 cm of length, 98 mg, and gray color). After decontamination with 5-min dichloromethane wash, strands were segmented and finely cut with scissors. 3 ng of methadone-D3 (internal standard) were added to 10 mg of sample, followed by incubation 12 h at 55 °C in methanol. After evaporation, each dry residue was dissolved in 100 μL of mobile phase (water + 0.2% ammonium hydroxide) in order to quantitate AMI and NOR by LC-MS/MS. 50 μL of sample was injected, an online sample clean-up was performed on an Oasis HLB column (Waters ® ), and the chromatographic separation was performed on an analytical column, Atlantis ® dC18 3 μm 2.1x150 mm (Waters ® ). Quantitation was performed using positive ionization mode on an API4000 (ABSciex ® ) and was based on two monitored transitions : 278,2 m/z -> 91 m/z; 278,2 m/z -> 105,1 m/z and 264,3 m/z -> 233,1 m/z ; 264,3 m/z -> 91,1 m/z, for AMI and NOR respectively. The cut off level was fixed at 0.01 ng/mg for AMI and NOR. Results For both parents all hair segments tested negative. For the baby, the results are summarized in the following table: Strand at To (without decontamination) Strand at T 5 weeks (with decontamination) Segment number Distance/root (cm) AMI NOR Segment number Distance/root (cm) AMI NOR (ng/mg) (ng/mg) 1 0 at 0.5 6.9 8.1 1 0 at 0.7 1.2 3.6 2 0.5 at 1.0 6.6 7.1 2 0.7 at 1.2 1.4 4.0 3 1.0 at 2.5 9.0 9.0 3 1.2 at 1.9 1.2 3.3 4 2.5 a 4.0 9.7 8.9 4 1.9 at 2.5 0.79 2.3 5 2.5 at 3.0 0.65 1.8 6 3.0 at 3.6 0.59 1.5 7 3.6 at 5 0.55 1.3 Conclusion High concentrations in all segments of the first strand of the child were in accordance with recent exposure and potential contamination by sweet. Maximal concentrations in segment number 2 of the second strand confirmed the acute intoxication. The presence of AMI and NOR in all segments of the second strand provided the proof of chronic exposure during several months before hair collection with regular increase. The profile of increasing concentrations revealed by segmentation allows the exclusion of in utero exposure.

HPTLC method for simultaneous determination of amlodipine besylate and enalapril maleate in pharmaceutical formulation

A rapid and simple high performance thin layer chromatography (HPTLC) method with densitometry at ?= 223 nm was developed and validated for simultaneous determination of Amlodipine and Enalapril from pharmaceutical preparation. Separation was performed on aluminum-backed silica gel 60F 254 HPTLC plates as stationary phase and using a mobile phase comprising of Toluene: Isopropanol: Glacial acetic acid (GAA): Methanol (6: 2: 0.6: 0.5 v/v/v/v) and 20 ml of mobile phase was used per chromatography run. After development, plates were observed under UV light. The validated lowest limit of detection was 80ng/spotand 200ng/spot whereas lowest limit of quantification was 150ng/spot and 400ng/spotfor Amlodipine and Enalapril respectively. The percentage assay of Amlodipine and Enalapril was found between 99.74 to 100.66% and 99.89 to 100.42% respectively. The described method has the advantage of being rapid and easy. Hence it can be applied for routine quality control analysis of Amlodipine and Enalapril from pharmaceutical preparation and stability studies.

Low-Pressure Chromatographic Separation of p-Hydroxybenzoates Using Sequential Injection with Lab-on-Valve System and Miniature Monolithic Column

The use of a small guard column (5-mm length) as a miniature analytical column in low-pressure liquid chromatography based on a sequential injection with lab-on-valve system was demonstrated. A strong initial mobile phase was used to rapidly deliver the sample zone to the column prior to simultaneous separation of analytes with weaker mobile phase. Separation of methyl-, ethyl-, propyl-, and butyl-4-hydroxybenzoates (MP, EP, PP, and BP) was achieved within about 120 s with sufficient peak resolution (all higher than 1.3) using 2,500 µL of mobile phase (composed of 1,087 µL of MeOH) per run. Detection limits were found to be 6.5, 8.0, 0.5, and 0.6 μmol L−1 for MP, EP, PP, and BP, respectively. The system was tested with common commercially available skin lotions and wet wipe products. The analysis results were in good agreement with those obtained from the high-performance liquid chromatography with a 10-cm-length packed column.

Liquid Chromatography Tandem Mass Spectrometry Method for Determination of Anti-Diabetic Drug Repaglinide in Human Plasma

A highly sensitive, accurate and rapid analytical method based on reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (RP-LC-ESI-MS/MS) has been developed and validated for the determination of repaglinide in human plasma using cetirizine as an internal standard (IS). The method was validated over a linear range of 0.5 - 100 ng/ml. After addition of IS, analytes were extracted from the plasma samples by liquid-liquid extraction using tert-butyl methyl ether as. The dried residue was reconstituted with 500 μL of mobile phase consisting of water/methanol/acetonitrile (62.5:20:17.5, v/v/v) and 0.2% formic acid. Chromatographic separations were achieved on a C18 analytical column. The analytes were detected with a triple quadrupole mass spectrometer using turbo V? ion spray source with positive ionization in multiple reaction monitoring (MRM) mode using MRM transitions m/z 453.3 > 162.2 and m/z 389.0 > 201.1 for the drug and IS, respectively. The proposed method was fully validated in terms of linearity, accuracy, precision, specificity, sensitivity, recovery and stability, giving results within the acceptable range. This method was successfully applied for a large number of authentic human plasma samples from a bioequivalence study.

Determination of immunoglobulin G in mare colostrum by high-performance gel permeation chromatography

A direct high-performance gel permeation chromatographic (HPGPC) method for the determination of immunoglobulin G in mare colostrum was established. HPGPC separation was performed on a TOSOH TSK-G4000PW(XL) column (300 mm x 7.8 mm, 5 microm) with 0.05 mol/L phosphate buffer solution (pH 6.9) as the mobile phase at a flow rate of 0.8 mL/min, and the column temperature was maintained at 25 degrees C. The injection volume was 20 microL. At the detection wavelength of 280 nm, the linear range was from 0.2 to 3.0 g/L (r2 = 0.999 5) with a detection limit of 0.08 mg/L (S/N = 10). The recovery was 97.47% with a relative standard deviation (RSD) of 1.22%. The RSDs of the peak area of stability, accuracy and reproducibility for the established method were 2.86%, 1.62% and 1.82%, respectively. Mare milk was collected from Zhaosu (China), a complete collection was stored in an ice box, then sent to a laboratory and stored in a low temperature refrigerator. The whey milk was prepared by centrifugation two times at 12 000 r/min and 4 degrees C for 30 min. The whey protein was obtained from the middle layer. A 2 mL volume of the whey milk was mixed with 23 mL of mobile phase. The average contents of IgG were from 35.0 g/L to 50.0 g/L at the first lactation (2 h), and the average contents of IgG were from 2.0 g/L to 4.0 g/L after 72 h. The relatively simple analytical method was proved to be accurate and precise in its application to mare colostrum.

Estimation of Pharmacokinetics of Propofol in Indian Pateints by HPLC Method

A quick and sensitive reversed phase high performance liquid chromatography (HPLC) method has been developed in Indian surgical patients to determine the concentration of Propofol In human plasma. Propofol can be isolated from human plasma by adding 1ml precipitating solution which consists of acetonitrile and perchloric acid (67:33) mixture, which also contains dibutylpthalate (1mg/ml) as an internal standard. The sample is mixed for two minute on a vortexer. The plasma substance precipitated by acetonitrile and perchloric acid are further separated by centrifugation. The supernatant is directly injected into the HPLC system with the help of autosampler. The analysis was carried out using column 250 × 4.6 mm column packed with10-μm Spherisorb reversed phase octadecyl silane particles (C 18 ). The 500ml of mobile phase (67:33:0.04) consisted of 335ml of acetonitrile and 165ml of distilled water and 200μl of acetic acid maintaining the pH 4.0.The flow rate of the mobile phase was 1.5ml/ min. propofol was monitored by a UV detector at a 270nm wavelength. The limit of detection of propofol (in human plasma) was found to be 0.0001μg/ml while limit of quantification was found to be 0.001μg/ml for a 20ul injection volume.

Two-column Sequential Injection Chromatography—New approach for fast and effective analysis and its comparison with gradient elution chromatography

This work presents novel approach in low-pressure chromatography flow systems—two-column Sequential Injection Chromatography (2-C SIC) and its comparison with gradient elution chromatography on the same instrument. The system was equipped with two different chromatographic columns (connected to selection valve in parallel design) for isocratic separation and determination of all components in composed anti-inflammatory pharmaceutical preparation (tablets). The sample was first injected on the first column of length 30 mm where less retained analytes were separated and then the sample was injected on the second column of length 10 mm where more retained analytes were separated. The SIC system was based on a commercial SIChrom™ manifold (8-port high-pressure selection valve and medium-pressure syringe pump with 4 mL reservoir) (FIAlab ®, USA) with two commercially available monolithic columns the “first column” Chromolith ® Flash RP-18e (25 mm × 4.6 mm i.d. with guard column 5 mm × 4.6 mm i.d.) and the “second column” Chromolith ® RP-18e (10 mm × 4.6 mm i.d.) and CCD UV–vis detector USB 4000 with micro-volume 1.0 cm Z flow cell. Two mobile phases were used for analysis (one for each column). The mobile phase 1 used for elution of paracetamol, caffeine and salicylic acid (internal standard) was acetonitrile/water (10:90, v/v, the water part of pH 3.5 adjusted with acetic acid), flow rate was 0.9 mL min −1 (volume 3.0 mL of mobile phase per analysis). The mobile phase 2 used for elution of propyphenazone was acetonitrile/water (30:70, v/v); flow rate was 1.2 mL min −1 (volume 1.5 mL of mobile phase per analysis). Absorbance was monitored at 210 nm. Samples were prepared by dissolving of one tablet in 30% acetonitrile and 10 μL of filtered supernatant was injected on each column (2 × 10 μL). The chromatographic resolution between all compounds was >1.45 and analysis time was 5.5 min under the optimal conditions. Limits of detection were determined at 0.4 μg mL −1 for paracetamol, at 0.5 μg mL −1 for caffeine and at 0.7 μg mL −1 for propyphenazone. The new two-column chromatographic set-up developed as an alternative approach to gradient elution chromatography shows evident advantages (time and solvent reduction more than one-third) as compared with single-column gradient SIC method with Chromolith ® Flash RP-18 (25 mm × 4.6 mm i.d. with guard column 5 mm × 4.6 mm i.d.).

Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders

Quantification of pristanic acid, phytanic acid, and very long chain fatty acids (i.e., hexacosanoic, tetracosanoic, and docosanoic acids) in plasma is the primary method for investigateing a multitude of peroxisomal disorders (PDs). Typically based on GC-MS, existing methods are time-consuming and laborious. In this paper, we present a rapid and specific liquid chromatography tandem mass spectrometric method based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was undertaken to improve the poor mass spectrometric properties of these fatty acids. Analytes in plasma (20 mul) were hydrolyzed, extracted, and derivatized with DAABD-AE in approximately 2 h. Derivatives were separated on a reverse-phase column and detected by positive-ion electrospray ionization tandem mass spectrometry with a 5 min injection-to-injection time. Calibration plots were linear over ranges that cover physiological and pathological concentrations. Intraday (n = 12) and interday (n = 10) variations at low and high concentrations were less than 9.2%. Reference intervals in normal plasma (n = 250) were established for each compound and were in agreement with the literature. Using specimens from patients with established diagnosis (n = 20), various PDs were reliably detected. In conclusion, this method allows for the detection of at least nine PDs in a 5 min analytical run. Furthermore, this derivatization approach is potentially applicable to other disease markers carrying the carboxylic group.

Determination of pesticides fenoxycarb and permethrin by sequential injection chromatography using miniaturized monolithic column

Sequential injection chromatography system equipped with miniaturized 10 mm monolithic column was used for fast simultaneous determination of two pesticides—fenoxycarb (FC) and permethrin (PM). The system was composed of a commercial sequential injection analysis (SIA) system (FIAlab ® 3000, 6-port selection valve and 5.0 mL syringe pump), commercially available column Chromolith™ RP-18e (10 mm × 4.6 mm i.d.) (Merck ®, Germany) and CCD UV–vis detector (USB 2000, Ocean-optics) with 1.0 cm Z-flow cell, absorbance was monitored at 225 nm. The mobile phase used for analysis was acetonitrile/water (60:40, v/v), flow rates were 0.6 mL min −1 for elution of fenoxycarb and 1.2 mL min −1 for elution of permethrin. For each analysis 4.8 mL of mobile phase was used. The chromatographic resolution between both compounds was >8 and analysis time was <6.5 min under the optimal conditions. Limits of detection were determined at 2.0 μg mL −1 for fenoxycarb and 1.0 μg mL −1 for permethrin. Samples were prepared by diluting with mobile phase and injected volume was 10 μL for each analysis. Developed method was applied to analysis of both pesticides in veterinary pharmaceutical foams and sprays ARPALIT ® Neo (Aveflor, Czech Republic). SIC method was compared with validated method (HPLC, reverse phase 100 mm monolithic column, gradient elution).

Proteomic analysis of platelet alpha-granules using mass spectrometry.

Platelets have three major types of secretory organelles: lysosomes, dense granules, and alpha-granules. alpha-Granules contain several adhesive proteins involved in hemostasis, as well as glycoproteins involved in inflammation, wound healing, and cell-matrix interactions. This article represents the first effort to define the platelet alpha-granule proteome using mass spectrometry (MS). We prepared a subcellular fraction enriched in intact alpha-granules from human platelets using sucrose gradient ultracentrifugation. alpha-Granule proteins were separated and identified using sodium dodecylsulfate polyacrylamide gel electrophoresis and liquid chromatography-tandem MS. In the sucrose fraction enriched in alpha-granules, we identified 284 non-redundant proteins, 44 of which appear to be new alpha-granule proteins, on the basis of a literature review. Immunoelectron microscopy confirmed the presence of Scamp2, APLP2, ESAM and LAMA5 in platelet alpha-granules for the first time. We identified 65% of the same proteins that were detected in the platelet releasate (J. A. Coppinger et al. [Blood 2004;103: 2096-104]) as well as additional soluble and membrane proteins. Our method provides a suitable tool for analyzing the granule proteome of patients with storage pool deficiencies.