Column-switching Liquid Chromatography Research Articles

Vacuum differential mobility spectrometry combined with column-switching liquid chromatography- mass spectrometry for the analysis of pyrrolizidine alkaloids in tea samples

The benefit of combining liquid chromatography (LC), supercritical fluid chromatography (SFC) and vacuum Differential Mobility Spectrometry – Mass Spectrometry (vDMS-MS) was investigated for the analysis of fourteen diastereomeric pyrrolizidine alkaloids (PA); intermedine, echinatine, lycopsamine, indicine, intermedine-N-oxide, echinatine-N-oxide, indicine-N-oxide, lycopsamine-N-oxide, senecivernine, senecionine, jacobine, senecivernine-N-oxide, senecionine-N-oxide, retrorsine. The mobile phase composition (15–100% MeOH and ACN), flow rate (8–100 µL/min), vDMS cell pressure, and F value showed an effect on the mobility behavior of the analytes. At 15% MeOH with a flow rate of 100 µL/min and 33 mbar vDMS pressure, 8 out 14 PA could be partially or totally separated by vDMS-MS. As well as providing an additional separation dimension vDMS improved the selectivity and a 5-minute assay method was developed for the quantification of 10 out of 14 single diastereomeric PA in tea samples, using a short LC column-switching and hyphenated to vDMS-MS in the selected ion monitoring mode. The performance of the method was found to be comparable with a 12-minute standard LC-MS/MS method using detection in the selected reaction monitoring mode. Additionally, the combination of vDMS and SFC-MS was investigated and suggests that the mixture of CO2/MeOH influences the CV shifting of the PA to more negative compensation voltage, and the signal-to-noise ratio is improved by a factor of three compared to SFC-MS without vDMS.

Recent advances in column switching high-performance liquid chromatography for bioanalysis

Bioanalysis is a relevant area of analytical chemistry for clinical studies. Biological samples are complex and diverse, so sample preparation represents a challenge when chromatographic methods are developed. According to the principles of green analytical chemistry (GAC), recent trends in sample preparation include miniaturization, automation (online coupling to the analytical instrument), and high-throughput performance. In this context, column switching liquid chromatography stands out as a multidimensional chromatographic method in which an extraction column is directly coupled to high-performance liquid chromatography (HPLC) systems. This online method consists of two steps and involves two columns, the extraction and the chromatographic columns. In the former column, the analytes are isolated from the sample and preconcentrated; in the latter column, the analytes are separated. Online systems improve the sensitivity and accuracy of analytical methods, consume lower amounts of organic solvents, and minimize sample handling. This review summarizes state-of-the-art column switching liquid chromatography and focuses on selective stationary phases for preconcentration of analytes (first dimension), including reversed phases, monolithic phases, restricted access materials (RAMs), and molecularly imprinted polymers (MIP). Principles, instrumental aspects, applications in bioanalysis, and future trends in column switching liquid chromatography are also discussed.

Quantification of biotin in plasma samples by column switching liquid chromatography – tandem mass spectrometry

Biotin (or Vitamin B7) is a vitamin where deficiency can be caused by inadequate intake. Biotin deficiency is rare, as most people get enough biotin from diet, since many foods contain biotin. In addition to biotin from food, intestinal bacteria can synthesize biotin, which can then be absorbed by the body. Supplementation with biotin has been advocated, mainly due to proposed beneficial effects on skin, nail and hair growth. There is no evidence that high biotin intakes are toxic, but a high intake may interfere with diagnostic assays that use biotin-streptavidin technology. These tests are commonly used to measure plasma concentrations of a wide range of hormones. Erroneous results may lead to misdiagnosis of various endocrine disorders. Supplementation with high-dose biotin has been used experimental for the treatment of diseases (e.g. multiple sclerosis) and high doses are used to obtain effect on nail and hair growth. On this background a demand for tests to determine if there is a risk of obtaining false test results when using biotin-streptavidin based tests have appeared. In this paper we present a method based on column switching liquid chromatography tandem mass spectrometry for the quantification of biotin in plasma and serum and explore the effects of biotin on an immunoassay based on biotin strept(avidin) chemistry.

Simultaneous steroids measurement in dogs with hyperadrenocorticism using a column-switching liquid chromatography-tandem mass spectrometry method.

We developed an analytical method using an on-line column-switching liquid chromatography with triple quadrupole mass spectrometry (LC/MS/MS) for quantifying multiple steroids in serum. Using the developed method, we evaluated the serum concentration of nine steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 21-deoxycortisol, deoxycorticosterone, progesterone, 17α-OH-progesterone and aldosterone) in dogs with hyperadrenocorticism (HAC). Serum was mixed with stable isotope internal standards and thereafter purified by the automated column-switching system. The limit of detection ranged 2–16 pg/ml for nine steroids. In the baseline samples, five steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, and 17α-OH-progesterone) were detected in all dogs. The concentrations of cortisone, 11-deoxycortisol, and 17α-OH-progesterone in dogs with HAC (n=19) were significantly higher those in dogs without HAC (n=15, P<0.02). After the adrenocorticotropic hormone stimulation test, six steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 17α-OH-progesterone, and deoxycorticosterone) were above the limit of quantification in all dogs. Cortisol, corticosterone, cortisone, and deoxycorticosterone concentrations of dogs with HAC were significantly higher than those of dogs without HAC (P<0.02). In addition, 11-deoxycortisol and 17α-OH-progesterone concentration was higher in dogs with HAC than in dogs without HAC (P=0.044 and P=0.048, respectively). The on-line column-switching LC/MS/MS would be feasible for measuring multiple steroids in dog serum. The results suggest that cortisone, 11-deoxycortisol, and 17α-OH-progesterone would be related to HAC. Further studies are warranted to assess the clinical feasibility of steroid profile in dogs with HAC.

Determination of epoxide impurity in sarpogrelate hydrochloride intermediate by UHPLC and column-switching liquid chromatography

The determination of genotoxic impurities, which is closely related to toxicological concern and daily dose, plays a key role in drug quality control. Epoxide impurity is a kind of genotoxic impurity with an epoxy ring structure during the synthesis process of sarpogrelate hydrochloride. According to the sarpogrelate hydrochloride daily dose, epoxide impurity is limited to the under 5 ppm level. The liquid chromatographic-tandem mass spectrometric (LC/MS/MS) or the gas chromatography-mass spectrometric (GC/MS) method is commonly used to characterize the epoxide impurity of sarpogrelate hydrochloride intermediates. However, these methods are not simple or economical enough to detect epoxide impurity. In this study, we resolved the problem by using the most common UV method with two ideas: one was to improve the absolute sensitivity, and the other was to reduce matrix effects. Both ultra high-performance liquid chromatography (UHPLC with high sensitivity LightPipe™ flow cells) and column-switching liquid chromatography methods were developed and validated for the quantitative determination of epoxide impurity in sarpogrelate hydrochloride intermediates. The limits of detection (LODs) of the UHPLC and column-switching liquid chromatography methods were 0.09 ppm (0.09 μg/g) and 0.33 ppm (0.33 μg/g), and the recovery rates of both methods were 87.2%–132.1% and 97.4%–100.1%, respectively. Both methods established and provided guidance for analysts to develop procedures for impurity control, especially for structures of impurity with similar matrices.

A novel, simple and robust method for the analysis of sulfonamides in fish farming water samples using column switching liquid chromatography

ABSTRACTA novel method for the online extraction and preconcentration of four sulfonamides was developed using column switching liquid chromatography. Sulfadiazine, sulfathiazole, sulfamethoxypyridazine and sulfamethoxazole were analysed in water samples and preconcentrated in a C18 guard column. Suitable validation parameters were obtained, such as precision, accuracy and relative recovery, in accordance with the validation guidelines of the Food and Drug Administration. Low limits of detection (0.05–0.09 µg L−1) and quantification (0.30 µg L−1, for all of them) were obtained. The quadratic polynomial model was used to adjust the calibration data, and the coefficients of determination were higher than 0.999 for all the analytes. The method was shown to be robust to the assayed parameters according to Youden’s test. The proposed method was successfully used to determine sulfonamides in 11 different fish farming water samples, in which sulfadiazine (0.732 µg L−1), sulfamethoxazole (0.531 µg L−1), sulfathiazole (0.546–1.856 µg L−1) and sulfamethoxypyridazine (0.369–1.509 µg L−1) were found.

Online extraction of antihypertensive drugs and their metabolites from untreated human serum samples using restricted access carbon nanotubes in a column switching liquid chromatography system

A novel analytical method was developed to determine 5 antihypertensive drugs of different pharmacological classes (angiotensin-converting enzyme inhibitors, calcium channel blockers, α-2 adrenergic receptor agonists, angiotensin II receptor blockers, and aldosterone receptor antagonists) and some of their metabolites in human serum. The untreated samples were directly analyzed in a column switching system using an extraction column packed with restricted access carbon nanotubes (RACNTs) in an ultra-high performance liquid chromatography coupled to a mass spectrometer (UHPLC–MS/MS). The RACNTs column was able to exclude approximately 100% of proteins from the samples in 2.0min, maintaining the same performance for about 300 analytical cycles. The method was validated in accordance with Food and Drug Administration (FDA) guidelines, being linear for all the determined analytes in their respective analytical ranges (coefficients of determination higher than 0.99) with limits of detection (LODs) and quantification (LOQs) ranging from 0.09 to 10.85μgL−1 and from 0.30 to 36.17μgL−1, respectively. High recovery values (88–112%) were obtained as well as suitable results for inter and intra-assay accuracy and precision. The method provided an analytical frequency of 5 samples per hour, including the sample preparation and separation/detection steps. The validated method was successfully used to analyze human serum samples of patients undergoing treatment with antihypertensive drugs, being useful for pharmacometabolomic, pharmacogenomic, and pharmacokinetic studies.

Simultaneous determination of phytochemicals and low-weight organic acids in Sophora japonica bud and premium noni juice through column-switching liquid chromatography connected with ion chromatography system

ABSTRACTIn this work, a liquid chromatography connected with an ion chromatography by the column-switching technique was developed for the simultaneous determination of three phytochemicals (scopoletin, rutin, and quercetin), three anions (chloride, sulfate, and phosphoric), and seven organic acids (lactic, acetic, propionic, formic, malic, oxalic, and citric acid) in Chinese traditional herbs, S. japonica bud and premium noni juice. A concentration column was used to collect organic acids and anions, and then the concentrated analytes were eluted to an analytical column by column-switching technique in which target analytes could be separated successfully and detected simultaneously within 70 min. This method showed good linearity with the linear correlation coefficients in the range of 0.9971–1.0000. The relative standard deviations of the peak areas were all less than 5.63% (n = 6). The limits of detection (LOD, S/N = 3) of anions and organic acids were in the range of 0.0006–0.0857 mg/L, whereas phytochemicals were in the range of 0.0123–0.0197 mg/L. Recoveries were in the range of 86.53–117.01% for the spiked sample. This method was successfully applied to the determination of the low-weight organic acids and phytochemicals in Chinese traditional herbal medicines, S. japonica bud and premium noni juice.

Characterization and application of restricted access carbon nanotubes in online extraction of anticonvulsant drugs from plasma samples followed by liquid chromatography analysis.

Anticonvulsant drugs are often used in the treatment of epilepsy. However, their therapeutic monitoring is often necessary in order to obtain an appropriate dose adjustment, due to the proximity between their therapeutic and toxic ranges. The aim of this study was to carry out the synthesis, characterization and use of restricted access carbon nanotubes (RACNTs) in an online method for the analyses of phenobarbital and carbamazepine and primidone from untreated human blood plasma by column switching liquid chromatography. Therefore, the synthesis of RACNTs was carried out through coating commercial Carbon nanotubes with bovine serum albumin (BSA) to subsequently use them as adsorbents in a column switching system operating in the backflush mode. This material was evaluated through the construction of the kinetic and isotherm curves. The experimental data for the interaction of primidone with RACNTs were adequately adjusted to the chemisorption and Sips models for the kinetic and adsorption studies, respectively. The analytical curves ranged from 2.0 to 40.0mgL-1, with correlation coefficients higher than 0.99, for all the analytes. The LODs of 0.1, 0.1 and 0.01μgmL-1 were defined for PHB, PRM and CBZ, respectively. The relative standard deviation values ranged from 1.0% to 8.4% for the intra assay precision and from 2.7% to 7.6% for inter assay precision. The relative error values ranged from -13.4% to 7.7% for the intra assay accuracy and from -8.6% to 2.5% for the inter assay accuracy. The method was adequately used in the therapeutic monitoring of anticonvulsant drugs in human plasma samples.

Direct extraction of tetracyclines from bovine milk using restricted access carbon nanotubes in a column switching liquid chromatography system.

This paper describes, for the first time, the use of restricted access carbon nanotubes (RACNTs) in the analysis of tetracyclines from milk samples, in a multidimensional liquid chromatographic system. Milk samples were initially acidified and centrifuged, and then the supernatant was directly analyzed in a column switching system in backflush configuration employing an extraction column of RACNTs. The sorbent was able to exclude all the remained proteins in less than 2.0min. The method was linear from 50 to 200μgL-1 and the coefficients of determination (r2) were 0.997, 0.992, 0.994 and 0.998 for oxytetracycline (OXI), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DOX), respectively. The analytical range included the maximum residue limits established by the regulatory agency.

Identification of Impurities in 5-Aminolevulinic Acid by Two-Dimensional Column-Switching Liquid Chromatography Coupled with Linear Ion Trap Mass Spectrometry

Identification of impurities in 5-aminolevulinic acid (ALA) by mass spectrometry is difficult, because MS-incompatible mobile phases, such as phosphate buffers or ion-pair reagents, need to be used to separate the major component from impurities. In this study, the unknown impurities in ALA have been identified by two-dimensional (2D) column-switching high-performance liquid chromatography (HPLC) coupled with linear ion trap mass spectrometry (LIT MS). The first-dimensional analytical column was a Gemini C18 (150 mm × 4.6 mm, 5 μm) with a non-volatile salt mobile phase at a flow rate of 1.0 mL min−1, and the second-dimensional analytical column was a ZORBAX SB C8 (150 mm × 4.6 mm, 3.5 μm) with a volatile salt mobile phase at a flow rate of 1.0 mL min−1. The detection wavelength was 205 nm. Mass spectra were acquired with an ESI source, in both positive and negative ion modes. Six impurities were identified by their MS2 and MS3 fragments, and the mass fragmentation patterns and structural assignments of these impurities were studied. The results obtained by the two-dimensional column-switching method were further compared with those of the conventional one-dimensional normal-phase HPLC–MS using an amide column and an MS-compatible mobile phase for separation. The two-dimensional column-switching method described herein proved to be advantageous in terms of the number of impurities identified. The column-switching and online demineralization technique made the mobile phase conditions compatible with mass spectrometry. Thus, the method solves the problem of incompatibility between non-volatile salt mobile phases and mass spectrometry, making it worthy of popularization and application in impurity identification.

Determination of Drugs by Online Column-Switching Liquid Chromatography

A fully automated and validated 2D online solid-phase extraction (SPE) high-performance liquid chromatographic method was described for the direct simultaneous determination of clenbuterol (CLEN) and doxazosin (DOX) in spiked human plasma samples. Chromatographic separation of the analytes was achieved by column-switching. SPE column packed with restricted access material for depleting matrix and a reversed phase HPLC column for eluting analytes were used with UV detection on 250 nm. A 10 mM acetate buffer (pH: 4) and acetonitrile mixture was used in gradient elution with 1 mL/min flow rate, as mass spectrometry friendly mobile phase. Calibration graphs were prepared for spiked plasma, and good linearity was achieved over a dynamic range of 75.0 ng/mL-200.0 µg/mL for CLEN and 37.5 ng/mL-100.0 µg/mL for DOX. Detection and quantification limits were 8.47 and 11.94 ng/mL, 25.66 and 36.18 ng/mL for CLEN and DOX, respectively. Online column-switching liquid chromatography technique combines the sample preparation by online SPE and separation steps in one allowing direct determination of drugs in biological fluids. This article could be assessed as a good example of combination of online sample preparation and separation, thus simultaneous determination of drugs in plasma samples.

Simultaneous determination of ten anticoagulant rodenticides in tissues by column-switching UHPLC-ESI-MS/MS.

This paper describes the development of a method for the simultaneous determination of ten anticoagulant rodenticides (coumafuryl, warfarin, pindone, coumatetralyl, coumachlor, difenacoum, bromadiolone, brodifacoum, chlorophacinone and flocoumafen) in the liver and kidney based on column-switching liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry. The simple sample preparation includes extraction with methanol. A C18 trapping column was used for online solid-phase extraction before analytical separation with the mobile phase comprising a mixture of 0.1% formic acid in water, methanol and acetonitrile. Chromatographic separation was achieved using a Thermo Hypersil ultra high-performance liquid chromatography (UHPLC) C18 column with the mobile phase consisting of 5 mM ammonium formate buffer (pH = 9) and methanol. The column-switching procedure ensured no matrix effects during electrospray ionization (ESI). Extraction recoveries ranged between 91 and 100% for liver and between 89 and 97% for kidney. The method showed good linearity up to 750 ng g(-1). The limit of detection ranged between 0.001 and 0.022 ng g(-1) for liver and between 0.001 and 0.028 ng g(-1) for kidney. The developed method was successfully used in several animal poisoning cases.

Determination of lansoprazole enantiomers in dog plasma by column-switching liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study.

Lansoprazole, a selective proton pump inhibitor, has a chiral benzimidazole sulfoxide structure and is used for the treatment of gastric acid hypersecretory related diseases. To investigate its stereoselective pharmacokinetics, a column-switching liquid chromatography with tandem mass spectrometry method was developed for the determination of lansoprazole enantiomers in dog plasma using (+)-pantoprazole as an internal standard. After a simple protein precipitation procedure with acetonitrile, matrix components left behind after sample preparation were further eliminated from the sample by reversed-phase chromatography on a C18 column. The fluent was fed to a chiral column for the separation of lansoprazole enantiomers. Baseline separation of lansoprazole enantiomers was achieved on a Chiralcel OZ-RH column using acetonitrile/0.1% formic acid in water (35:65, v/v) as the mobile phase at 40°C. The linearity of the calibration curves ranged from 3 to 800 ng/mL for each enantiomer. Intra- and inter-day precisions ranged from 2.1 to 7.3% with an accuracy of ±1.7% for (+)-lansoprazole, and from 1.6 to 6.9% with an accuracy of ±3.5% for (-)-lansoprazole, respectively. The validated method was successfully applied for the stereoselective pharmacokinetic study of lansoprazole in beagle dog after intravenous infusion.

Discovery of xanthine oxidase inhibitors from a complex mixture using an online, restricted-access material coupled with column-switching liquid chromatography with a diode-array detection system

To find potential lead compounds for antigout drug discovery, an automated online, restricted-access material coupled with column-switching liquid chromatography with a diode-array detection (RAM-LC-DAD) system was developed for screening of xanthine oxidase (XO) inhibitors and their affinity rankings in complex mixtures. The system was first evaluated by analyzing a mixture of six compounds with known inhibition of XO. Nonspecific binding to the denatured XO was investigated and used as the control for screening. Subsequently, the newly developed system was applied to screening of a natural product, Oroxylum indicum extract, and four compounds which could specifically interact with XO were found and identified as oroxinB, oroxinA, baicalin, and baicalein. The results were verified by a competitive binding test using the known competitive inhibitor allopurinol and were further validated by an inhibition assay in vitro. The online RAM-LC-DAD system developed was shown to be a simple and effective strategy for the rapid screening of bioactive compounds from a complex mixture.

Quantitative determination of 11-nor-9-carboxy-tetrahydrocannabinol in hair by column switching LC–ESI-MS3

Hair analysis has been regarded as an alternative method to urine analysis in forensic and criminal cases. Cannabis (marijuana) is one of the most widely used drugs in the world and it has been controlled in South Korea since 1976. Identification of 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) in hair can be an important proof of cannabis use because it can exclude the possibility of passive cannabis smoke exposure. In this study, we described a quantitative method of THCCOOH in hair using simple liquid-liquid extraction (LLE), selective column switching liquid chromatography with electrospray ionization (ESI)-MS(3). For the column switching system three columns (precolumn, trap column and analytical column) were used. Valve switch from the precolumn to the trap column was set from 3.0 to 4.0 min because THCCOOH appeared around 3.5 min with this precolumn. After 4.0 min the valve was switched to the original position and the analytes in the trap column were eluted onto the analytical column. Resolution occurred in this column and eluted into the ESI-MS(3) system. The internal standard was THCCOOH-d3. We used ESI-negative-MS(3) transition of ions at m/z 343 to 299 to 245 (343/299/245) and m/z 346 to 302 to 248 (346/302/248) for quantification of THCCOOH and THCCOOH-d3, respectively. The validation results of selectivity, matrix effect, recovery, linearity, precision and accuracy, and processed sample stability were satisfactory. The limit of detection (LOD) was 0.05 pg/mg and the limit of quantification (LOQ) was 0.10 pg/mg. The range of concentration of THCCOOH from 98 authentic human hair was 0.13-15.75 pg/mg. This method was successfully applied in the analysis of authentic human hair samples.

Quantification of achiral and chiral methylsulfonyl polychlorinated biphenyl metabolites by column-switching liquid chromatography–atmospheric pressure photoionization–tandem mass spectrometry

An enantioselective heart-cut column-switching liquid chromatography–atmospheric pressure photoionization–tandem mass spectrometry method was developed for the analysis of 25 methylsulfonyl polychlorinated biphenyl metabolites in tissue extracts. Use of a pyrenyl-ethyl silica column in the first dimension enabled separation of all but two pairs of isobaric analytes. Enantioseparation was achieved for 9 out of the 10 atropisomeric analytes using a Chiralpak AD-H amylose-based column within 93min, resulting in greater chromatographic resolution of enantioseparation over shorter analysis time by up to a factor of three, compared to previous one-dimensional and multi-dimensional gas chromatography-based methods. Precision for concentration and enantiomer fraction measurements was within 11% and 3% relative standard deviation, respectively. Limits of detection ranged from 0.01 to 1.73ng on-column. Meta-congeners had poorer sensitivity (i.e., ng on-column), consistent with existing gas chromatography-based methods. Despite this limitation, the method was successfully applied to the analysis of Greenland sledge dog adipose tissue extracts, which had highly non-racemic residues of 4-methylsulfonyl-2,2′,3,5′,6-pentachlorobiphenyl and 4′-methylsulfonyl-2,2′,3,3′,4,6′-hexachlorobiphenyl, consistent with past reports in Arctic mammals.

A column-switching liquid chromatography–tandem mass spectrometry method for quantitation of 2-cyanoethylmercapturic acid and 2-hydroxyethylmercapturic acid in Chinese smokers

The acrylonitrile metabolites 2-cyanoethylmercapturic acid (CEMA) and 2-hydroxyethylmercapturic acid (HEMA) have been determined in human urine using an automated column-switching procedure. A diluted sample was centrifuged just prior to being injected into a reusable precolumn packed with a restricted access material and coupled to a liquid chromatography–tandem mass spectrometry system. This method achieved satisfactory reproducibility and accuracy. Average intra- and interday variations (% relative standard deviations) ranged from 2.4 to 3.8% for CEMA and from 2.7 to 10.5% for HEMA. The limits of quantification were 0.003 and 0.099ng/ml for CEMA and HEMA, respectively. It was used to study the uptake of acrylonitrile from smoke constituents by both nonsmokers and smokers of different tar yield cigarettes under ISO 3308 smoking condition. Metabolite concentrations in smoker urine samples were approximately 12 times higher compared with those in nonsmokers for CEMA and 3 times higher for HEMA. Urinary CEMA levels show a clear dose–response relationship with daily cigarette consumption and urinary cotinine. CEMA can also discriminate between smokers of different ISO cigarettes. Because HEMA is not specific, it is only slightly related to smoking and acrylonitrile exposure. The validated biomarker CEMA will continue to be useful for studies of acrylonitrile uptake by smokers.

Method development and validation of the simultaneous determination of a novel topoisomerase 1 inhibitor, the prodrug, and the active metabolite in human plasma using column-switching LC–MS/MS, and its application in a clinical trial

A robust and sensitive method using liquid chromatography–tandem mass spectrometry was developed and validated for the simultaneous determination of a novel topoisomerase 1 inhibitor CH0793076 (3076), the prodrug CH4556300 (TP300), and the active metabolite CH0793011 (3011) in human plasma. All plasma analyzed with this method was acidified with 1 M HCl and 46% citric acid solution in a ratio of 100:10:1 (v:v:v) to avoid the pH-based degradation of TP300 and to shift the equilibria of 3076 and 3011 between the lactone and carboxylate forms towards the lactone forms. After the plasma proteins were precipitated with methanol:acetonitrile:HCl 1 M (50:50:1, v:v:v) containing stable isotopic internal standards, the analytes were trapped on an Xterra MS C18 column (10 × 2.1 mm i.d., 5 μm) and separated on a Gemini C18 column (50 × 2.0 mm i.d., 5 μm) using column-switching liquid chromatography. Electrospray ionization in the positive-ion mode and multiple reaction monitoring were used to quantify the analytes with transitions m/ z 587.2 > 441.2 for TP300, 459.1 > 415.2 for 3076, and 475.1 > 361.1 for 3011. The inter- and intra-day precisions were below 12%, and the accuracy was between −16% and 16% at the lower limit of quantitation (LLOQ) and between −11% and 14% at the other quality controls. The LLOQs of TP300, 3076, and 3011 were 0.8, 0.04, and 0.04 ng/mL, respectively. The validated method was successfully applied to clinical sample analysis and incurred sample reanalysis was also conducted.

Quantification of Fentanyl in Human Serum by Column-Switching Liquid Chromatography and Tandem Mass Spectrometry

Analytical methods for pharmacokinetic studies with quantification of the highly potent anaesthetic drug fentanyl in serum must be rugged, sensitive, precise and accurate. To address these analytical demands and facilitate an automated approach to sample clean-up, a new method was developed for human serum using back-flush columnswitching and high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). Prior to injection, protein precipitation in trichloroacetic acid was performed in each sample. Samples were diluted with internal standard (D5-fentanyl) in 10% (w/v) trichloroacetic acid, kept at 4oC for 20 min and centrifuged followed by injection of 40μL supernatant onto a BioTrap 500 MS extraction column. Using a time programmed six-port valve switch, the extracted drug was back-flushed onto a Zorbax SB-Aq analytical column, gradient eluted and finally detected after electrospray ionisation with multiple reaction monitoring (MRM) of the transitions m/z 337 → m/z 188 and m/z 342 → m/z 188 for fentanyl and D5-fentanyl, respectively. Mean inter-assay accuracy (n=5) ranged from 93 to 101 % and inter-assay precision (n=5) ranged from 3.7 to 9.4% (C.V.). The method was used in a preliminary study of 4 patients after application of transdermal patches and found fully applicable for future pharmacokinetic studies. This is the first on-line, column-switching LC-MS/MS method validated and demonstrated suitable for quantification of fentanyl in human serum.