LC APCI MS Research Articles

Analysis of Hexanal and Heptanal in Human Blood by Simultaneous Derivatization and Dispersive Liquid–Liquid Microextraction then LC–APCI–MS–MS

A new analytical approach, simultaneous derivatization and dispersive liquid–liquid microextraction followed by liquid chromatography–atmospheric-pressure chemical ionization tandem mass spectrometry, has been developed for analysis of hexanal and heptanal in human blood. In the derivatization and extraction procedure a solution of 2,4-dinitrophenylhydrazine (derivatization reagent) in 85 μL acetonitrile (dispersive solvent) and 50 μL tetrachloromethane (extraction solvent) was rapidly injected into the aqueous sample containing hexanal and heptanal. Within a few seconds the aldehydes were derivatized and simultaneously extracted. After centrifugation, the hydrazones in the sediment phase were analyzed by LC–APCI–MS–MS. Derivatization and extraction conditions were investigated systematically. Under the optimum conditions enrichment factors for hexanal and heptanal in a 1-mL sample were 63 and 73, respectively. The calibration plots were linear in the ranges 0.5–100 and 100–1,000 nmol L−1, respectively, and the respective limits of detection (LOD) were 0.17 and 0.076 nmol L−1. Reproducibility and recovery were good. The experimental results were compared with those obtained by use of solid-phase extraction and polymer monolithic microextraction. Because sample derivatization, extraction, and concentration were combined in a single step, the proposed method enabled simple, rapid, inexpensive, and efficient analysis of aldehydes in blood. The method has great potential for clinical analysis of biologically relevant aldehydes.

Curcuminoid Biosynthesis by Two Type III Polyketide Synthases in the Herb Curcuma longa

Curcuminoids found in the rhizome of turmeric, Curcuma longa, possess various biological activities. Despite much attention regarding the biosynthesis of curcuminoids because of their pharmaceutically important properties and biosynthetically intriguing structures, no enzyme systems have been elucidated. Here we propose a pathway for curcuminoid biosynthesis in the herb C. longa, which includes two novel type III polyketide synthases. One of the type III polyketide synthases, named diketide-CoA synthase (DCS), catalyzed the formation of feruloyldiketide-CoA by condensing feruloyl-CoA and malonyl-CoA. The other, named curcumin synthase (CURS), catalyzed the in vitro formation of curcuminoids from cinnamoyldiketide-N-acetylcysteamine (a mimic of the CoA ester) and feruloyl-CoA. Co-incubation of DCS and CURS in the presence of feruloyl-CoA and malonyl-CoA yielded curcumin at high efficiency, although CURS itself possessed low activity for the synthesis of curcumin from feruloyl-CoA and malonyl-CoA. These findings thus revealed the curcumin biosynthetic route in turmeric, in which DCS synthesizes feruloyldiketide-CoA, and CURS then converts the diketide-CoA esters into a curcuminoid scaffold.

Determination of food emulsifiers in commercial additives and food products by liquid chromatography/atmospheric-pressure chemical ionisation mass spectrometry

A new, reliable liquid chromatography/atmospheric-pressure chemical ionisation mass spectrometry (LC–APCI-MS) method was developed for the quantitative determination of food emulsifiers composed of mono- and diacylglycerols of fatty acids (E471 series) in complex food matrices. These additives are extremely interesting for the food industry because of their useful properties. Indeed, they improve the manufacture of products by acting as foams and creams stabilisers, crumb-softeners, or antistaling agents. The proposed method also allows us to qualitatively characterise new food emulsifiers composed of other acid esters of mono- and diacylglycerols (E472 series). The validation of the method was performed on blank minicake spiked samples for detection limits (reaching ppm levels), linearity, recovery, precision, and accuracy. The method was then successfully applied to commercial additives containing mixtures of emulsifiers, as well as to food products such as margarines and minicakes.

Dopant assisted-atmospheric pressure photoionization (DA-APPI) liquid chromatography–mass spectrometry for the quantification of 27-hydroxycholesterol in plasma

27-Hydroxycholesterol (27OH-Chol) is of potential diagnostic interest due to its role in maintaining whole-body cholesterol homeostasis. Dopant assisted-atmospheric pressure photoionization (DA-APPI) has improved the sensitivity of 27OH-Chol analysis, in comparison to the published LC-APCI–MS method, allowing quantification from a very low amount of sample (≤50 μL plasma). The method was validated for quantification from 50 μL and 15 μL plasma, with the limit of quantification (LOQ) of 10 ng/mL and 40 ng/mL plasma, respectively. A further advantage is that no prior derivatization was needed, unlike the LC-ESI–MS or the standard GC–MS method. The method validation also resulted in good linearity and recovery of 91–106%. The within-day and between-day coefficient of variation were less than 15% while giving the accuracy of 90.9–113.4%. This report summarizes the effects of some critical parameters on the ionization of 27OH-Chol using APPI, as well as the validation of the method. The sensitivity achieved with DA-APPI broadens the usefulness of the LC–MS method in clinical applications.

Selective, sensitive and simple LC–APCI–MS method for the quantitation of Alpha-Tocopheryl Nicotinate in human plasma

A sensitive and specific method using liquid chromatography with atmospheric pressure chemical ionization mass spectrometry (LC–APCI–MS) has been developed and validated for the identification and quantification of Alpha-Tocopheryl Nicotinate in human plasma. A simple liquid–liquid extraction procedure was followed by injection of the extracts onto a C 18 column with isocratic elution and detection using a single quadrupole mass spectrometer in selected ion monitoring (SIM) mode. The method was tested using six different plasma batches. Linearity was established for the concentration range 2–1500 ng/ml, with a coefficient of determination ( r) of 0.9998 and good back-calculated accuracy and precision. The intra- and inter-day precision values were less than 15% and the deviations were within ± 5.0%. The lower limit of determination was reproducible at 0.5ng/ml with 0.5 ml plasma. The proposed method enables the unambiguous identification and quantification of Alpha-Tocopheryl Nicotinate for pre-clinical and clinical studies.

Cryo-irradiation as a terminal method for the sterilization of drug aqueous solutions

The aim of this study is to evaluate the specificities of the irradiation of drugs in frozen aqueous solution. The structures of the degradation products were determined to gain insight into the radiolysis mechanisms occurring in frozen aqueous solutions. Metoclopramide hydrochloride and metoprolol tartrate were chosen as models. The frozen solutions were irradiated at dry ice temperature by high energy electrons at various doses. The drug purity (chemical potency) and the radiolysis products were quantified by HPLC-DAD. Characterization of the degradation products was performed by LC-APCI-MS-MS. The structures of the radiolysis products detected in irradiated frozen aqueous solutions were compared to those detected in solid-state and aqueous solutions (previous studies). For both metoclopramide and metoprolol, solute loss upon irradiation of frozen aqueous solutions was negligible. Five radiolysis products present in traces were identified in irradiated metoclopramide frozen solutions. Three of them were previously identified in solid-state irradiated metoclopramide crystals. The two others were formed following reactions with the hydroxyl radical (indirect effect). Only one fragmentation product was observed in irradiated metoprolol frozen solutions. For both drugs, radiosterilization of frozen solutions, even at high doses (25 kGy), was found to be possible.

Neonatal death following clozapine self-poisoning in late pregnancy: An unusual case report

The report presents a fatal poisoning of a neonate occurring in the final stage of gestational life and evoked by his mother, who, while 9 months pregnant, took a toxic dose of clozapine aiming at committing suicide. She was also severely poisoned, but ultimately was saved. The woman had been taking the medication due to schizophrenia and depression prior to conception, and the discontinuation of the drug in the course of pregnancy increased the risk of the woman attempting suicide. In the course of comprehensive toxicological analysis based on the developed analytical procedure with the use of LC–APCI–MS, clozapine and its two metabolites, norclozapine and clozapine– N-oxide, were determined in postmortem blood, liver and kidney in concentrations explaining the death of the neonate. The interpretation of the above-described case is complex and – apart from toxicological aspects – also involves issues associated with psychiatry, pharmacotherapy in pregnancy and medicolegal problems.

Fatality due to the use of a designer drug MDMA (Ecstasy)

Drugs of abuse belonging to the amphetamine derivatives, which are often taken by adolescents and young adults, pose a serious risk associated with uncontrolled ingestion that sometimes leads to fatal outcomes. The number of deaths, however, related to Ecstasy is small when compared to the frequency of its use. The report presents a fatal poisoning with MDMA – Ecstasy of a 22-year-old male with a documented history of drug abuse. The observations of witnesses to the event made within the period between the exposition and fatal outcome may document the characteristic behavior of a person in the course of progressive poisoning. Toxicological investigations of the autopsy specimens carried out by means of liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry (LC–APCI–MS–MS) demonstrated the presence of MDMA and its metabolite MDA in the blood of the victim, and the concentration level justified the fatal outcome (MDMA – 1.42 mg/L, MDA – 0.17 mg/L), while the detection of high MDMA levels in a 6-cm long strand of hair separated into three segments (11.64 ng/mg in S 1; 8.74 ng/mg in the S 2; 15.51 ng/mg in the S 3) confirmed the history of drug abuse. The report describing the results of macro and microscopic examinations aiming at assessing internal organ damage suggested a mild hepatic damage.

Simultaneous Determination of Triptolide and Tripdiolide in Extract of Tripterygium wilfordii Hook. f. by LC–APCI-MS

A novel method using liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry in the negative selected ion monitoring mode has been developed and validated for rapid simultaneous determination of triptolide and tripdiolide in the extract of Tripterygium wilfordii Hook. f. The molecular ions m/z [M–H]− 359 and 375 were selected for the quantification in selected ion monitoring mode for triptolide and tripdiolide. Standard calibration curve was linear over the concentration range of 0.12–24 and 0.15–30 μg mL−1 for triptolide and tripdiolide. The relative standard deviations of intra- and inter-day were in the range of 4.7–9.9 and 8.9–12.6%. The average recoveries were between 96.4 and 104.6%. The limits of quantitation were 2.0 × 10−3 and 2.5 × 10−3 μg mL−1 for triptolide and tripdiolide.

Determination of tributyltin and 4-hydroxybutyldibutyltin chlorides in seawater by liquid chromatography with atmospheric pressure chemical ionization-mass spectrometry

A liquid chromatographic method is described for the simultaneous determination of tributyltin (TBT) and the hydroxylated intermediate 4-hydroxybutyldibutyltin (OHBuDBT). Separation was achieved in reverse phase mode on a cyanopropyl-bonded silica column under a gradient elution. Various organic solvents and additives were tested and the optimum composition of the mobile phase contained methanol, water, formic acid and tropolone as a complexing agent. Butyltin compounds were detected with an ion trap mass spectrometer interfaced to a liquid chromatograph with an atmospheric pressure chemical ionization source (LC–APCI-MS). Identification and fragmentation pattern of OHBuDBT chloride in full scan MS and MS/MS are reported for the first time using LC–APCI-MS. Gas chromatography–mass spectrometry (GC–MS) spectrum of the same compound is also reported for the first time for comparison purpose. This method allowed limits of detection (LOD) of 35 and 26 ng mL −1 for TBT and OHBuDBT, respectively, based on successive injections of 10 μL of blank seawater extract. A liquid–liquid extraction procedure using n-hexane–ethyl acetate was developed for the simultaneous analysis of TBT and OHBuDBT chlorides in natural seawater and allowed average recoveries from 72 to 96% for the two compounds at three different spiking levels.

Application of HPLC–APCI–MS with a C-30 reversed phase column for the characterization of carotenoid esters in mandarin essential oil

Liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (LC–APCI–MS) was used to identify carotenoid esters in mandarin essential oil. A gradient of methanol and methyl-t-butyl ether on a C-30 reversed-phase column was employed. The polymeric C-30 stationary phase has demonstrated superior resolution for carotenoid separation in comparison to the traditional C-18 column. Alkaline saponification is usually performed before carotenoid extract analysis by HPLC, thus no information concerning carotenoid esters in natural samples can be obtained. Because carotenoid esters are known to be more stable than free carotenoids, the main native carotenoid esters composition present in the non-volatile residue of mandarin essential oil was determined for the first time, thus providing a possible carotenoid esters profile characteristic for this oil. Moreover, good amounts of β-cryptoxanthin and its esters were determined, indicating mandarin peel as a potential source of vitamin A precursors. Copyright © 2005 John Wiley & Sons, Ltd.

Application of matrix solid phase dispersion to the determination of imidacloprid, carbaryl, aldicarb, and their main metabolites in honeybees by liquid chromatography–mass spectrometry detection

A method based on matrix solid phase dispersion (MSPD) using C18 as dispersant and dichloromethane–methanol as eluent and liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (LC–APCI–MS) has been developed for the simultaneous determination of imidacloprid, 6-chloronicotinic acid, carbaryl, aldicarb, aldicarb sulfoxide, and aldicarb sulfone in honeybees. The proposed method was compared with liquid–liquid extraction (LLE) combined with LC–APCI–MS analysis. Spiked blank samples were used as standards to counteract the matrix effect observed in the chromatographic determination. Recovery studies were performed at different fortification levels. Average recoveries by MSPD varied from 61% of 6-chloronicotinic acid to 99% of aldicarb sulfoxide and relative standard deviations were equal or lower than 14%. Limit of detections ranged from 0.004 mg kg −1 for imidacloprid to 0.09 mg kg −1 for 6-chloronicotinic acid. Results obtained by both methods were compared, MSPD showed higher recoveries and sensitivity than LLE for most pesticides, except for carbaryl. As MSPD is easier to perform, faster, consumes less sample and organic solvents than LLE, its application for pesticide analysis in honeybees is suggested.

Liquid chromatographic–mass spectrometric—analyses of anaerobe protein degradation products

A liquid chromatography–electrospray ionisation-mass spectrometry (LC–ESI-MS) method and a liquid chromatography–atmospheric pressure chemical ionisation-mass spectrometry (LC–APCI-MS) method was developed to identify metabolites from the anaerobe protein degradation to biogas. As consequence of a process failure the biogas production breaks down with increasing substrate loading, whereas different metabolites accumulate in the fermentation media. These compounds were identified as metabolites from the anaerobe degradation of the aromatic amino acids phenylalanine, tyrosine and tryptophan and accumulate in concentrations up to 300 mg/L when casein was used as model substrate.

Enzymic conversion of 3β-hydroxy-5-ene-steroids and their sulfates to 3-oxo-4-ene-steroids for increasing sensitivity in LC–APCI-MS

A method of increasing the sensitivity of 3β-hydroxy-5-ene (Δ 5)-steroids in liquid chromatography–atmospheric pressure chemical ionization-mass spectrometry (LC–APCI-MS) based on the structural conversion by cholesterol oxidase (ChO) was demonstrated. The Δ 5-steroids were rapidly converted to their 3-oxo-4-ene (Δ 4)-forms by the treatment with ChO and the obtained Δ 4-forms provided 3–14-fold higher sensitivity compared to intact steroids in the positive-APCI-MS. This enzymic conversion method was also applied to the sulfated conjugates of Δ 5-steroids after solvolysis. The method enabled the detection of trace levels of dehydroepiandrosterone and androstenediol 3-sulfate in human serum, which could not be detected by the usual LC–MS.

Quantitative determination and structural characterization of isoflavones in nutrition supplements by liquid chromatography–mass spectrometry

In this paper, an accurate and route method was developed to quantitative determine daidzein, genistein, glycitein, daidzin, glycitin, 6″- O-acetyldaidzin, 6″- O-acetylglycitin and 6″- O-acetylgenistin contents in selected high and low isoflavones in nutrition supplements by on line liquid chromatography–atmospheric pressure chemical ionisation mass spectrometry (LC–APCI-MS). Improved extraction and hydrolysis methods of the isoflavones from three nutrition supplements were also studied and a rapid extraction method was developed. Comparison of different MS 2 and MS 3 spectra of isoflavones and some unknown compounds were also explored and proposed pathway fragments of nine isoflavones were first systematically suggested.

Determination of the sum of malachite green and leucomalachite green in salmon muscle by liquid chromatography–atmospheric pressure chemical ionisation-mass spectrometry

A sensitive method for the determination and confirmation of the sum of malachite green (MG) and leucomalachite green (LMG) in salmon muscle has been developed. It is based on the use of an oxidative pre-column reaction which converts LMG into MG previous to liquid chromatography–atmospheric pressure chemical ionisation–mass spectrometry (LC–APCI–MS) analysis. The determination of both compounds together constitutes a good screening method to confirm the presence of this kind of residue, taking into account that the combined signals will provide a gain of sensitivity. The detection limit, determined for spiked salmon samples using the confirmatory ion m/ z 313, was 0.15 μg/kg. The recoveries determined at a spiking level of 2 μg/kg were 85 and 70% for LMG and MG, respectively, with respective relative standard deviations of 1.3 and 3.1%.

Screening for library-assisted identification and fully validated quantification of 22 beta-blockers in blood plasma by liquid chromatography–mass spectrometry with atmospheric pressure chemical ionization

A liquid chromatographic–mass spectrometric assay with atmospheric pressure chemical ionization (LC–APCI–MS) is presented for screening for, library-assisted identification (both in scan mode) and quantification (selected-ion mode) of the beta-blockers acebutolol, diacetolol, alprenolol, atenolol, betaxolol, bisoprolol, bupranolol, carazolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, propranolol, sotalol, talinolol and timolol in blood plasma after mixed-mode (HCX) solid-phase extraction (SPE) and separation by reverse-phase liquid chromatography with gradient elution. The validation data were within the required limits. The assay was successfully applied to authentic plasma samples allowing confirmation of diagnosis of overdose situations as well as monitoring of patients’ compliance.

Determination of dithiocarbamates and metabolites in plants by liquid chromatography–mass spectrometry

A quantitative matrix solid-phase dispersion and liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (LC–APCI–MS) method is outlined for the simultaneous analysis of dithiocarbamates (DTCs) and their degradation products in plants. Compounds analyzed are dazomet, disulfiram, thiram and the metabolites ethylenthiourea and propylenthiourea. The performance of two different sample preparation protocols, the proposed one and other based on solid-phase extraction, as well as, of both atmospheric pressure ionization sources, APCI and electrospray, were compared. The effect of several parameters on the extraction, separation and detection was studied. Dithiocarbamates and metabolites were dispersed with carbograph, eluted with a mixture of dichloromethane–methanol, and then, identified by monitoring the base peak of the spectra corresponding to [ M+H] +. The method was validated for avocados, cherries, lemons, nuts, oat, oranges, peaches, rice and tomatoes. Average recoveries varied from 33 to 109%, and relative standard deviation were between 4 and 21% with limits of quantification ranged from 0.25 to 2.5 mg kg −1, except for thiram and disulfiram, which were not recovered from fruits with high acid content. The procedure was applied to the determination of DTCs and their metabolites in fruits, vegetables and cereals taken from different markets of Valencia, Spain.

Simultaneous quantification of Vitamins A, D 3 and E in fortified infant formulae by liquid chromatography–mass spectrometry

A novel method for the simultaneous quantification of Vitamins A, D 3 and E in fortified infant formulae has been developed using isocratic normal-phase liquid chromatography with positive atmospheric pressure chemical ionization mass spectrometry (LC–APCI-MS). Food products were saponified and the vitamins were extracted by solid-phase extraction (SPE) on a Chromabond XTR ® cartridge. Quantification of Vitamins D 3 and E were performed with Vitamin D 2 and 5,7-dimethyltocol (DMT) as internal standards (IS), respectively while no IS was used for Vitamin A. Detection of the vitamins was made in the selected ion monitoring (SIM) mode. MS calibration curves were linear between 0.15 and 12 mg/l for Vitamin A, 5–400 μg/l for Vitamin D 3 and 0.25–20 mg/l for Vitamin E with regression coefficient r 2>0.996 and the limits of detection were below 1.4 ng. The repeatability (CV) obtained on a reference dietetic infant formula was 2.3% for Vitamin A, 2.6% for Vitamin E and 5.9% for Vitamin D 3. The between-day variations (CV) over 6 days were in the ranges of 2.4–6.9% for the three vitamins. The mean recoveries from a reference infant formula spiked with all three vitamins ranged from 96 to 105% with a relative standard error less than 9%. The applicability of the method was demonstrated by analyzing a set of infant formula and infant cereals; similar results were obtained with the LC–MS method and reference HPLC methods.

Application of high-performance liquid chromatography–mass spectrometry to detection of diuretics in human urine

A rapid, sensitive and reliable high-performance liquid chromatographic–mass spectrometric method for the detection of 25 diuretics in human urine has been developed. Atmosphere pressure chemical ionization (APCI) and electrospray ionization (ESI) modes were evaluated. A 2-ml volume of urine was extracted under basic conditions and separated on an Agilent Zorbax SB-C 18 column (150×2.1 mm, 5 μm). The mobile phase consisted of formic ammonium–formic acid buffer (pH 3.5) and acetonitrile. The effects of capillary temperature, sheath gas pressure and compositions of mobile phase on the sensitivity were studied. The recoveries of most of the diuretics were 75–95%. In the full scan mode, the limits of detection of the 25 diuretics were 0.25–25 ng/ml for APCI and 0.6–250 ng/ml for ESI. Under the optimal conditions, 14 diuretics from authentic urine samples were detected successfully by LC–APCI-MS. To obtain more fragmentation information on the chemical structure for positive confirmation, tandem mass analysis was also investigated.