Thermospray Liquid Chromatography-mass Spectrometry Research Articles

OO′-(1,4-xylylene) bispilocarpic acid esters as new potential double prodrugs of pilocarpine for improved ocular delivery. I. Synthesis and analysis

An analogue series representing novel diesters of bispilocarpic acid, O, O′-( l,4-xylylene) bispilocarpic acid esters, were synthesized as double prodrugs of pilocarpine in order to improve the ocular delivery characteristics of the drug. In previous studies, various bispilocarpic acid monoesters have been synthesized and evaluated as prodrugs of pilocarpine. However, these derivatives suffer from instability in aqueous solution and hence were not suitable as a pilocarpine prodrug. Based on earlier results, 1,4-xylylene bispilocarpate was selected as the starting material for the synthesis of diesters. Bispilocarpic acid diesters were prepared by esterifying the free hydroxyl groups of bispilocarpic acid monoesters. All the diesters formed a salt with 3 equivalents of fumaric acid. The yields of bispilocarpic acid diester fumarates varied from 48 to 99%. The identification of the compounds and evaluation of the purity of synthetic products were performed by liquid chromatography with UV detection, thermospray liquid chromatography mass spectrometry, electron impact ionization mass spectrometry and 1H-NMR spectroscopy. The elemental composition of each compound was determined on high-resolution mass spectrometry by measuring the accurate mass of the molecular ion.

Applications of thermospray liquid chromatography-mass spectrometry in photochemical studies of pesticides in water

Thermospray liquid chromatography-mass spectrometry (TSP-LC-MS) in the positive- and negative-ion modes (PI and NI, respectively) was used for the characterization of the pesticides aldicarb, carbaryl, cyanazine, fenitrothion, linuron and parathion-methyl, including the corresponding photodegradation products. The LC analyses were performed on RP-18 columns using methanol-water (70:30 or 50:50) + 0.05 M ammonium acetate or 0.05 M ammonium formate. Photodegradation studies of pesticides in distilled water were performed using a suntest apparatus, except for fenitrothion, for which a high-pressure mercury lamp was used because of slow photodegradation. The main photodegradation pathways corresponded to dealkylation, deamination, dehalogenation and hydroxylation. Photodegradation experiments were carried out using solutions of the different pesticides at 10–200 ppm in distilled water. For carbaryl photolysis is very slow in distilled water, so 5% of acetone was needed as photosensitizer. When PI-mode TSP-LC-MS was employed, aldicarb sulphoxide, monolinuron, hydroxysimazine and fenitrooxon could be identified as breakdown products of aldicarb, linuron, cyanazine and fenitrothion, respectively. In NI-mode TSP-LC-MS aldicarb sulphoxide, 1-naphthol and p-nitrophenol could be identified as the main degradation products of aldicarb, carbaryl and parathion-methyl, respectively. As all the pesticides were dissolved in methanol for solubility reasons, methoxy analogues of the degradation products of cyanazine were also identified. In the PI mode of operation the base peak generally corresponded to [M + H] + for cyanazine and its photodegradation products and to [M + NH 4] + for linuron, aldicarb and fenitrothion and their corresponding degradation products. For carbaryl [M + NH 4] + was also the base peak, but its main photodegradation product, 1-naphthol, could only be identified under NI conditions. In the NI mode of operation different processes such as proton abstraction, (dissociative) electron capture and anion attachment, take place. Fragment ions such as [M] ·− for aldicarb sulphoxide, the main photodegradation product of aldicarb, [M - H] − for 1-naphthol and [M - CONHCH 3] − for carbaryl and anion attachment ions corresponding to [M + CH 3COO] − for aldicarb sulphoxide and 1-naphthol and to [M - CONHCH 3 + CH 3COOH] − for carbaryl were formed. A tentative photodegradation pathway for the different pesticides in water is postulated.

Evaluation of eluents in thermospray liquid chromatography-mass spectrometry for identification and determination of pesticides in environmental samples

The influence of different eluents in positive and negative ion mode thermospray liquid chromatography-mass spectrometry was studied with several groups of pesticides, including carbamates, chlorotriazines, phenylureas, phenoxy acids and organophosphorus and quaternary ammonium compounds, and the corresponding degradation products. Using the positive ion mode in combination with reversed-phase eluents the base peaks generally corresponded either to [M + H] + for the chlorotriazines and their hydroxy metabolites or to [M + NH 4] + for the carbamates, the phenylureas, the organophosphorus pesticides and their oxygen analogues. In the negative ion mode different processes such as (dissociative) electron-capture and anion attachment mechanisms occurred. Fragment ions such as [M — CONHCH 3] − for the carbamates, [M − H] − for the chlorotriazines, phenylureas and chlorinated phenoxy acids and [M] . −, [M − R] − (R being a methyl or ethyl group) for organophosphorus pesticides were usually formed. Depending on the eluent additive used (ammonium acetate, ammonium formate and/or chloroacetonitrile), three different adduct ions were formed: [M + CH 3COO] −, [M + HCOO] − and [M + Cl] −. Normal-phase eluents with cyclohexane, n-hexane and/or dichloromethane provided more structural information and enhanced the response of several compounds. The positive ion mode was useful for the detection of chlorinated phenoxy acids and chlorophenols which could not be detected in the positive ion mode using reversed-phase systems. The base peaks generally corresponded to [M] . +, [M + H] + or [M − Cl] +. For the characterization of difenzoquat, a quaternary ammonium pesticide of which trace level analysis is troublesome, a post-column ion-pair extraction system was used. An aqueous mobile phase with a sulphonate-type counter ion was applied and an extraction solvent containing cyclohexane-dichloromethane- n-butanol (45:45:10) was used in thermospray liquid chromatography-mass spectrometry. Illustrative examples of the determination of residue levels of pesticides in soil matrices are shown.

Identification of by-products in phenylisocyanate pre-column derivatization by thermospray liquid chromatography-mass spectrometry

Thermospray liquid chromatography-mass spectrometry has been applied to the identification of by-products in precolumn derivatization by phenylisocyanate. These byproducts are eluted early in the same chromatographic region as the low molecular weight derivatives and were located by chromatographic analysis of a blank sample. Their identification would offer further qualitative information in the use of phenylisocyanate as a derivatizing agent. Five compounds resulted from the reaction of phenylisocyanate and the reaction medium were identified: two from a reaction between phenylisocyanate and methanol, two from the reaction between phenylisocyanate and water, and one from the polymerisation of phenylisocyanate.

Thermospray liquid chromatography-mass spectrometry for the characterization of sulphate ester conjugates

Formation of polar conjugates is a well documented metabolic pathway for xenobiotics containing phenolic hydroxyl groups. This paper describes the analysis of two sulphate ester conjugates by fast atom bombardment mass spectrometry anermospray liquid chromatography-mass spectrometry. Thermospray liquid chromatography-mass spectrometry proved the more succesful technique for obtaining the molecular weight of the intact conjugate, but only by removal of the buffer from the high-performance liquid chromatography eluent.

Use of thermospray liquid chromatography-mass spectrometry to aid in the identification of urinary metabolites of a novel antiepileptic drug, Lamotrigine

The use of thermospray liquid chromatography-mass spectrometry allowed the structural elucidation of a number of urinary metabolites of Lamotrigine, 3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine, formed after administering the drug to man, Cynomolgus monkey and rabbit. This data when combined with the data obtained from high-performance liquid chromatography with radiochemical detection enabled us to determine the types and amounts of unchanged drug and metabolites excreted in urine by man and a number of laboratory animal species. This technique was particularly useful as it highlighted a previously unknown fact that Lamotrigine is metabolised to form two different N-glucuronides, one of which is resistant to cleavage in vitro by a crude β-glucuronidase preparation from Helix pomatia.

Evaluation of an automated thermospray liquid chromatography-mass spectrometry system for quantitative use in bioanalytical chemistry

An automated thermospray liquid chromatography-mass spectrometry system is described, including an autosampler and a gradient liquid chromatography system controlled from the mass spectrometer data system. The performance and reliability of the equipment during unattended operation were evaluated by repeated injections of standard solutions of some antiasthmatic drugs, using deuterium-labelled analogues as internal standards. High sensitivity and reproducibility were achieved during a 19-hour run, incorporating gradient elution and a total of 54 injections. The relative standard deviation of the peak area measurement of the internal standards was in the range of 6.5–8.2%. The corticosteroid budesonide can be routinely measured in plasma down to 0.1 nmol/1. Direct injection of a small plasma volume into the thermospray liquid chromatography-mass spectrometry system could be used to monitor drug plasma levels during a toxicity study in dogs.

Increasing thermospray response for cortisol by derivatization

The 21-hydroxyl group of cortisol was selectively acetylated under mild conditions without affecting the more sterically hindered 11β- and 17α-hydroxyl groups. The reaction was performed with a mixture of acetic anhydride and triethylamine in acetonitrile and was complete in less than 15 min at room temperature. The thermospray mass spectrum of cortisol 21-acetate showed minimal fragmentation with the [M + H] + ion as the base peak. High sensitivity was achieved for acetylated cortisol during selected ion monitoring, the signal-to-noise ratio being increased by a factor of about 4, compared to underivatized cortisol. The limit of detection of cortisol 21-acetate was estimated at 0.24 pmol injected, making thermospray liquid chromatography-mass spectrometry competitive with gas chromatography-mass spectrometry for the determination of cortisol in biological fluids.

Development of analytical methods for some penicillins in bovine milk by ion-paired chromatography and confirmation by thermospray mass spectrometry

Analytical methods for the determination of cloxacillin, ampicillin/hetacillin, and amoxicillin in bovine milk were developed. The methods involved ultrafiltration of milk diluted with methanol, acetonitrile, and water on a 10,000-dalton cut-off filter. Separation of penicillins from other milk components was accomplished by ion-paired chromatography using a microbore column. The penicillins were detected using ultraviolet photodiode array (UV-PDA) detection and confirmed by thermospray liquid chromatography-mass spectrometry (LC-MS). The thermospray spectra of these compounds exhibited [M + H]+ and [M + Na]+ ions along with several fragment ions. The limits of detection for these antibiotics were estimated to be 50 to 100 ppb for LC with UV-PDA detection and 100-200 ppb for thermospray LC-MS detection.

High-performance liquid chromatography—thermospray mass spectrometry of prostaglandin and thromboxane acetyl derivatives

A quantitative analysis of prostaglandin-related substances has been developed. Hydroxyl groups of prostaglandin and thromboxane were acetylated by acetic anhydride, the mixture was partially purified on a Sep-Pak C 18 cartridge and analysed by high-performance liquid chromatography combined with thermospray mass spectrometry. Using this method, twenty kinds of prostaglandin derivative could be detected simultaneously within 11 min on a selected-ion monitoring detection chromatogram without a gradient system. Generally, the base ion, [M + H − n(60)] +, is produced through elimination of acetic acid ( n = number of the hydroxyl group of prostaglandin or thromboxane). The detection limit for these derivatives was ca. 0.2 pmol at the levels of prostaglandin-related substances prior to derivatization. They could be analysed in the range 0.5–10 pmol. The assay was successfully applied to prostaglandin-related substances in human seminal fluid and rat brain.

Mobile phase variations in thermospray liquid chromatography—mass spectrometry of pesticides

The effect of four different mobile phase compositions with reversed-phase methanol—water (50:50) + 0.05 M ammonium acetate, methanol—water (50:50) + 0.05 M ammonium formate, acetonitrile—water (50:50) + 0.05 M ammonium acetate and acetonitrile—water (50:50) + 0.05 M ammonium formate were compared in filament-on thermospray liquid chromatography—mass spectrometry for the determination of carbamate and chlorotriazine pesticides. In the positive-ion mode, [M + H] + and [M + NH 4] + were generally the base peaks for the chlorotriazines and the carbamates, respectively. Depending on the mobile phase used, other adduct ions obtained corresponded to [M + CH 3CN + H] +, [M + CH 3OH + NH 4] +, [M + CH 3COONH 4 + NH 4 − 2H 2O] +, [M + CH 3CN + NH 4] +, [M + CH 3COONH 4 + H − H 2O] + and the dimer [2M + H] +. In the negative-ion mode, [M − H] − and adducts with the ionizing additive [M + CH 3COO] − or [M + HCOO] − were obtained. Other ions for the carbamates carbaryl and oxamyl corresponded to [M − CONHCH 3 + CH 3COOH] − and [M − CON(CH 3) 2 + HCOO] −, respectively. The variation of mobile phase composition provides additional structural information in thermospray liquid chromatography—mass spectrometry with no appreciable loss of sensitivity. Applications are reported for the determination of carbamate and chlorotriazine pesticides at the ng/g level in spiked and real soil samples, respectively.

Detection of ribose-methylated nucleotides in enzymatic hydrolysates of RNA by thermospray liquid chromatography—mass spectrometry

A procedure has been developed for the detection and characterization of ribose-methylated dinucleotides of the type NmpN′ in enzymatic digests of RNA. Differences in reaction products from hydrolysis using RNase T 2, which will not cleave NmpN′, and hydrolysis by nuclease P 1 are analyzed by thermospray liquid chromatography—mass spectrometry. The method is applicable to dinucleotides present in nanogram range quantities and is suited for the characterization of new or unexpected ribose-methylated nucleotides for which chromatographic mobilities are not known.

Determination of the metabolites of terfenadine in human urine by thermospray liquid chromatography—mass spectrometry

Thermospray liquid chromatography-mass spectrometry (TSP LC-MS) was used to determine human urinary metabolites of terfenadine after oral administration of terfenadine tablets. In addition to the two previously identified major metabolites, azacyclonol (MDL 4829) and the 'acid' metabolite (MDL 16,455), three additional metabolites were also detected. One of the additional metabolites was identified as the 'alcohol' metabolite (MDL 17,523) and the other two were proposed to be an 'aldehyde' and a 'ketone-acid' metabolites from their TSP mass spectra. The results of this study demonstrated that TSP LC-MS is a useful technique for the study of terfenadine biotransformation.

Thermospray and particle beam liquid chromatographic—mass spectrometric analysis of coumarin anticoagulants

Positive ion mass spectra were obtained from several coumarin oral anticoagulants (phenprocoumon, warfarin, acenocoumarol and dicoumarol) and derivatives by liquid chromatography—thermospray mass spectrometry (LC—TSP-MS) and liquid chromatography—electron impact mass spectrometry (LC—EI-MS) to assess the use of LC—MS methods for the determination of these compounds in biological materials. LC—TSP mass spectra showed a single [M + 1] + ion with no fragmentation; LC—EI mass spectra showed fragment ions which were similar in mass and relative intensities to those obtained by conventional EI-MS. These data should serve as a basis for the development of LC—MS methods for the qualitative and quantitative analysis of coumarin anticoagulants in biological samples. LC—TSP-MS was applied to the determination of phenprocoumon in a plasma extract from an anticoagulated patient.

Synthesis and identification of pilocarpic acid diesters, prodrugs of pilocarpine

A series of new pilocarpic acid diesters were synthesized to obtain prodrugs for pilocarpine with varying physico-chemical properties. Thermospray liquid chromatography-mass spectrometry (TSP-LC-MS), liquid chromatography with UV-detection (LC-UV) and NMR-spectroscopy were used for the identification of the synthetic products and for evaluation of their purity including typical impurities (pilocarpic acid monoester, pilocarpine). TSP-LC-MS-analysis was performed in the reversed-phase mode using acetonitrile (60%)-0.2 M ammonium acetate (40%) as mobile phase. In LC-UV-analysis chromatographic separation was carried out on a reversed-phase column and the mobile phase consisted of methanol (71%) and 0.02 M potassium dihydrogen phosphate, pH 4.5 (29%). Electron ionization-mass spectrometry (EI-MS) was used for elucidation of structures. Elemental compositions of the substances were verified with high resolution-mass spectrometry (HR-MS). The complete establishment of structures presented was based on 1H-, and COSY-NMR-spectroscopy joined to TSP-LC-MS-analysis.

Application of liquid chromatography—thermospray mass spectrometry in the analysis of glycerophospholipid molecular species

We report the application of high-performance liquid chromatographic (HPLC) separation with ultraviolet detection and direct, on-line, structural analyses by mass spectrometry of glycerobenzoate derivatives from complex mixtures of phospholipid molecular species. Individual phospholipids were resolved from total lipid extracts by thin-layer chromatography (TLC). Diradylglycerols were released from phospholipids by phospholipase-C treatment, converted to diradyl glycerobenzoates and subsequently separated by TLC into subclasses (alk-1-enylacyl, alkylacyl and diacyl types). The molecular species within each subclass were resolved by HPLC with an octadecyl reversed-phase column in acetonitrile—isopropanol (80:20, v/v). Individual peaks were quantitated at the picomole level by measuring absorbance at 230 nm. After post-column addition of methanol—0.2 M ammonium acetate (50:50, v/v), peaks were introduced through the thermospray interface into a VG Masslab 30–250 quadrupole mass spectrometer. Molecular species showed as base peaks the salt adducts of the molecular ion which permitted easy deduction of the overall fatty acyl composition. In addition, the diglyceride fragment of each species was found at [MH — 122] + and two fragments formed by the loss of the fatty acyl groups (R) in the sn-1 or sn-2 position were found at [M — R 1] + and [M — R 2] +, respectively. Since preferential release of either fatty acyl group was observed in positional isomers, the ratio of the intensity of these fragments gave information on the position of the fatty acyl groups in the individual HPLC peaks. We show that the use of on-line mass spectrometry, however, provides easy identification of all molecular species present in a complex phospholipid mixture, even when more than one molecular species is contained in an HPLC peak.

Daily Cortisol Production Rate in Man Determined by Stable Isotope Dilution/Mass Spectrometry

Growth retardation as well as the development of Cushingoid features in adrenally insufficient patients treated with the currently accepted replacement dose of cortisol (33-41 mumol/day.m2; 12-15 mg/m2.day) prompted us to reevaluate the cortisol production rate (FPR) in normal subjects and patients with Cushing's syndrome, using a recently developed thermospray liquid chromatography-mass spectrometry method. The stable isotope [9,12,12-2H3]cortisol was infused continuously for 31 h at about 5% of the anticipated FPR. Blood samples were obtained at 20-min intervals for 24 h, spun, and pooled in 4-h groups. Tracer dilution in plasma was determined by liquid chromatography/mass spectrometry. The method was validated with controlled infusions in 6 patients with adrenal insufficiency. Results from 12 normal volunteers revealed a FPR of 27.3 +/- 7.5 mumol/day (9.9 +/- 2.7 mg/day) or 15.7 mumol/day.m2; 5.7 mg/m2. day). A previously unreported circadian variation in FPR was observed. Patients with Cushing's syndrome demonstrated unequivocal elevation of FPR and cortisol concentration correlated during each sample period in normal volunteers, indicating that cortisol secretion, rather than metabolism, is mainly responsible for changes in plasma cortisol. Our data suggest that the FPR in normal subjects may be lower than previously believed.

Hydroxylation and glucoside conjugation in the microbial metabolism of the diterpene sclareol

1. The microbial metabolism of sclareol (1), a labdane diterpene ditertiary alcohol, was studied. Preliminary screening identified a number of microorganisms capable of metabolizing sclareol. 2. Preparative scale fermentation of growing cultures of Bacillus cereus UI-1477 resulted in the production of seven metabolites which have been characterized as 3 beta-hydroxysclareol (2), 2 alpha-hydroxysclareol (3), 18-hydroxysclareol (4), 2 alpha,18-dihydroxysclareol (6), 8 alpha,13 beta-dihydroxy-labd-14-en-3 beta-O-beta-D-glucoside (5), 8 alpha,13 beta-dihydroxy-labd-14-en-18-O-beta-D-glucoside (7), and 8 alpha,13 beta-dihydroxy-labd-14-en-2 alpha-O-beta-D-glucoside (10) by chemical, enzymic, and spectral data, especially 2D-n.m.r. techniques and thermospray liquid chromatography-mass spectrometry analysis.

Reversed-phase high-performance liquid chromatography-thermospray mass spectrometry of alprenolol and its ketoxime analogues

Thermospray mass spectrometric detection is applied for alprenolol and its isomeric ( E and Z) ketoxime analogues separated by reversed-phase high-performance liquid chromatography. The thermospray process results in high-intensity [M+H] + ion formation. This method of detection provides high level of molecular specificity, and offers advantages for the identification of the stereoisomeric oximes due to the unique fragmentation pattern of the spectra.

Determination of O-ethyl S-2-diisopropylaminoethyl methylphosphonothioate (VX) by thermospray liquid chromatography—mass spectrometry

The determination of the nerve agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothioate (VX) by thermospray liquid chromatography—mass spectrometry was studied. The solvent system acetonitrile—methanol—0.25 M ammonium acetate was used on a reversed-phase C 18 column. By selected ion monitoring at the protonated molecular ion of VX ( m/z 268), the predominant peak in its thermospray mass spectrum, an amount of 200 pg could be detected. For the determination of VX in water at levels below 1 ng/ml, preconcentration by C 18 cartridges was investigated. The applicability of the method was demonstrated by the determination of VX in spiked river waters. A concentration of 0.1 ng/ml could be detected starting from a water sample of 50 ml. A second application concerned the analysis of water extracts of spiked soil samples.