Identification of pesticides by liquid chromatography--particle beam mass spectrometry using electron ionization and chemical ionization

Liquid chromatography-mass spectrometry (LC-MS) with a particle beam (PB) interface is used to separate and identify a group of pesticides. The mass spectra obtained under the different ionization modes, electron ionization (EI) and positive and negative chemical ionization (PCI and NCI) are compared. The operating conditions under each mode, determined by studying the influence on the ion abundance of the ion source temperature of the EI mode, and the gas pressure and ion source temperature in the methane CI were optimized. EI was more sensitive than PCI and NCI and of the latter two modes, NCI gave higher responses, especially for organophosphorus compounds. When on-line solid-phase extraction-LC-PB-MS was applied to real samples, limits of detection in full scan mode were in the range of 0.5 and 10 micrograms l-1 for EI. The analysis of real samples by on-line solid-phase extraction-LC-PB-MS enabled EI detection of one of the pesticides studied and confirmation by PCI and NCI. The combined EI/CI information also enabled the detection of some non-target compounds.     

Molecular species of sphingomyelin: determination by high-performance liquid chromatography/mass spectrometry with electrospray and high-performance liquid chromatography/tandem mass spectrometry with atmospheric pressure chemical ionization

In a sphingomyelin-enriched sample of polar lipids from bovine milk, molecular species of intact sphingomyelin were separated by normal-phase high-performance liquid chromatography and detected by mass spectrometry (MS) for structural information. First, by using electrospray with positive ionization (ESI), protonated molecules ([M + H]+) were detected. Second, in atmospheric pressure chemical ionisation (APCI+), in-source fragmentation of sphingomyelin ions led to the formation of ceramide ions. With the ceramide ions as precursors, ions representative of both the long-chain base (LCB) parts and the fatty acid (FA) parts were detected in APCI-MS/MS via collision-induced decomposition (CID). Using this procedure, it was possible to determine the sphingomyelin molecular masses using ESI+ and then their respective LCB-FA combinations(s) using APCI+(-)MS/MS. At least 36 protonated molecules of intact sphingomyelin were detected in the bovine milk sample. The combinations found covered a range of molecular masses from 673 to 815 Da. The 12 most common protonated molecules (constituting approximately 90% of the total ion current in ESI) were composed of at least 25 different LCB-FA combinations. Saturated and unsaturated LCBs and FAs were detected in addition to hydroxy fatty acids. The most common LCBs were 16:1, 17:1, 18:1 and 19:1, whereas the most common FAs were 16:0, 22:0, 23:0 and 24:0. LCB-FA combinations of sphingomyelin from bovine brian, bovine erythrocytes and chicken egg yolk are also presented.     

Electron impact and chemical ionization mass spectrometry of steroidal spirolactones

Electron impact and chemical ionization mass spectra are reported for several steroidal spirolactones and their TMS ethers. The electron impact spectra were characterized generally by low abundance molecular ions and large numbers of fragment ions. Methane chemical ionization spectra exhibited high intensity [M+H]+ and/or [M+H-H2O]+ or [M+H-TMSOH]+ ions with few other fragment ions. Ammonia chemical ionization spectra had intense [M+H]+ and/or [M+NH4]+ ions with a few fragment ions generally formed by loss of H2O or TMSOH from these parent ions. Ammonia chemical ionization gave intense parent ions even for polyhydroxy compounds and their TMS ethers in contrast to methane chemical ionization. The results of this study suggest that a combination of electron impact with ammonia chemical ionization mass spectrometry would offer the best techniques for detection and identification of these compounds in biological fluids.     

High-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry for the analysis of 1,2,3,4-tetrahydro-beta-carboline derivatives

Zinc has been shown to have antioxidant properties and to exhibit inhibitory effects on apoptosis. In this work we investigated the effect of zinc on DNA integrity and on apoptosis of HaCaT keratinocytes. Cells were submitted to zinc deprivation by a diffusible zinc chelator, (N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine) (TPEN) or supplied with zinc chloride and submitted to UVB radiation. After cell exposure to TPEN for 2 h, strand breaks significantly impaired DNA resistance to alkaline denaturation. DNA strand breaks induced by a 6 h TPEN application were significantly prevented if zinc chloride was supplied together with the chelator. TPEN also generated, after 4-6 h of application, cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes), features of cell death by apoptosis. Moreover, UVB irradiation led to early DNA strand breaks and to an increase in cytoplasmic nucleosomes which was maximum 10 h after irradiation. These effects were prevented by the supply of zinc chloride (0.1 mM) in the culture medium. These results suggest that zinc ions interfere with the apoptosis process at an early stage, by decreasing DNA damage able to trigger apoptosis.     

Analysis of malachite green and metabolites in fish using liquid chromatography atmospheric pressure chemical ionization mass spectrometry

Malachite green (MG), a traditional agent used in aquaculture, is structurally related to other carcinogenic triphenylmethane dyes. Although MG is not approved for use in aquaculture, its low cost and high efficacy make illicit use likely. We developed sensitive and specific methods for determination of MG and its principal metabolite, leucoMG (LMG), in edible fish tissues using isotope dilution liquid chromatography atmosphere pressure chemical ionization mass spectrometry. MG and LMG concentrations were measured in filets from catfish treated with MG under putative use conditions (ca. 250 and 1000 ppb, respectively) and from commercial trout samples (0-3 and 0-96 ppb, respectively). Concentrations of LMG in edible fish tissues always exceeded those of MG. A rapid cone voltage switching acquisition procedure was used to simultaneously produce molecular ions for quantification and diagnostic fragment ions for confirmation of MG and metabolites. The accurate and precise agreement between diagnostic ion intensity ratios produced by LMG in authentic standards and incurred fish samples was used to unambiguously confirm the presence of LMG in edible fish tissue. This suggested the validity of using LMG as a marker residue for regulatory determination of MG misuse. Additional metabolites derived from oxidative metabolism of MG or LMG (demethylation and N-oxygenation) were identified in catfish and trout filets, including a primary arylamine which is structurally related to known carcinogens. The ability to simultaneously quantify residues of MG and LMG, and to confirm the chemical structure of a marker residue by using LC/MS, suggests that this procedure may be useful in monitoring the food supply for the unauthorized use of MG in aquaculture.     

High-performance liquid chromatography with electrospray ionization mass spectrometry and diode array ultraviolet detection in the identification of flavonol aglycones and glycosides in berries

High-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) was used to study flavonol aglycones and glycosides in berries. For the identification of aglycones, photodiode-array detection (DAD) was also used. The HPLC-ESI-MS technique is highly valuable in the identification of flavonol aglycones and glycosides from berry extracts. This ionization technique provides information on the structure of the aglycones and glycosides without time-consuming pre-purification or derivatization steps. Quercetin aglycone was identified with both ESI-MS and DAD in all of the berries studied. Myricetin aglycone was identified with both techniques in three berries. Hexose, deoxyhexose-hexose and pentose derivatives of quercetin were the most abundant flavonol glycosides identified. Two glycosides of myricetin and one glycoside of kaempferol were identified in blackcurrant. To confirm the data obtained using the HPLC-ESI-MS procedure, fractions of the glycosides from four berries were separated, hydrolyzed, silylated and the sugars were analyzed using gas chromatography-mass spectrometry.     

Hair analysis for nordiazepam and oxazepam by gas chromatography--negative-ion chemical ionization mass spectrometry

The sub-Tenon's technique uses blunt dissection and a blunt probe to inject local anaesthetic into the posterior sub-Tenon's space. This avoids the potentially catastrophic complications which result from passing a sharp needle blindly into the orbit and retrobulbar space. The anatomy of Tenon's capsule and the block technique is described. Results of the block quality and degree of patient comfort from 300 consecutive sub-Tenon's blocks are also described. No significant complications occurred in this series. Single-quadrant sub-Tenon's block offers an excellent quality of anaesthesia, is virtually painless to perform and avoids complications due to passage of a sharp needle into the orbit.     

High-performance liquid chromatographic determination of L-aspartyl-L-phenylalanine methyl ester in various food products and formulations

A simple, rapid, and specific high-performance liquid chromatographic (HPLC) procedure is described for the analysis of the chemical sweetener L-aspartyl-L-phenylalanine methyl ester (aspartame). Using a strong cation exchange column and pressures less than 1000 psig, an analysis can be performed in less than 15 min. The technique has been applied to a wide range of food products and formulations. No interferences were found in the samples studied. Recoveries are quantitative, and the coefficients of variation for replicate analyses are less than or equal to 2.5%.     

High-performance liquid chromatographic determination of a new oral thrombin inhibitor in the blood of rats and dogs

Donovanosis is a genital ulcer disease that occasionally has extragenital manifestations. This report describes a case of disseminated donovanosis in a 54-year-old woman from northern Australia who had subsequent thoracic vertebral osteomyelitis and spinal cord compression. Malignancy and vertebral tuberculosis were the major differential diagnoses. The patient had no genital lesions at the time of diagnosis of extragenital donovanosis but had undergone a hysterectomy, thus raising the possibility of prior disease of the uterine cervix (most previous cases have been associated with primary cervical disease). Despite treatment with doxycycline, she had no significant neurological improvement. Donovanosis disseminated to bone has been reported in 18 cases in the last 55 years. Awareness of donovanosis in the differential diagnosis of osteomyelitis and prompt pelvic examinations enabling early diagnosis of occult cervical disease are the most important measures in preventing morbidity and mortality due to disseminated donovanosis.     

Quantitative measurement of a new synthetic hetrazepine derivative, BN50730, in human plasma and urine by combined liquid chromatography-negative chemical ionization mass spectrometry using a particle beam interface

A new simple and sensitive assay has been developed for the quantitative measurement of BN50730 at the picomole level in human plasma and urine. The drug and the internal standard (BN50765) were measured by combined liquid chromatography-negative chemical ionization mass spectrometry with methane as the reagent gas. A simple solid-liquid extraction procedure was used to isolate BN50730 from complex biological matrices. Mild operating conditions were required to assay the parent drug with a particle beam interface from Hewlett-Packard. The mass spectrometer was tuned to monitor the intense ion m/z 333, which was generated in the ion source by a dissociative capture process. This assay was performed with 1 ml of plasma or 0.1 ml of urine, and the quantification limit of the method was statistically calculated as 1 ng ml-1. The very low relative standard deviation and mean percentage of error calculated during the different within-day or between-day repeatability assays clearly demonstrate the ruggedness of the technique for the routine determination of BN50730 in the biological fluids. Some preliminary results on the pharmacokinetics of the drug are presented to illustrate the applicability of this new powerful LC-MS method.     

Reverse-phase capillary high performance liquid chromatography/high performance electrospray ionization mass spectrometry: an essential tool for the characterization of complex glycoprotein digests

The B-domain of recombinant human Factor VIII comprises 909 amino acids and is extensively N- and O-glycosylated, in that at least 20 different sites are occupied by numerous carbohydrate structures. This domain was incubated with trypsin and subjected to liquid chromatography electrospray ionization mass spectrometry analysis, using an electrospray orthogonal acceleration time-of-flight mass spectrometer as the detector for a capillary reversed phase HPLC separation of the digest. The inherent high mass resolution afforded by this instrument provides both ion charge state determination and high accuracy mass measurement that are of significant advantage in defining such highly complex mixtures.     

Determination of four anabolic steroid metabolites by gas chromatography/mass spectrometry with negative ion chemical ionization and tandem mass spectrometry

A gas chromatography/mass spectrometry method is described which uses negative ion chemical ionization and tandem mass spectrometry for the determination of anabolic steroid metabolites. Four anabolic steroid metabolites to be derivatized by Pentafluoropropionic anhydride (PFPA) were determined using gas chromatography/mass spectrometry (GC/MS) with negative chemical ionization (NCI) and NCI/MS/MS. The repeatability and reproducibility of this procedure were in the range of 5.3-9.7% and 6.1-10.2%, respectively. This method of derivatization with PFPA for NCI and NCI/MS/MS was useful to determine four metabolites of nandrolone, dromostanolone, methenolone and boldenone. The derivatized metabolites of boldenone could be detected to 2 ppb and the other three steroids could be detected to 25 ppb in urine at a signal-to-noise ratio of S/N = 3.     

Application of high-performance liquid chromatograph-electrospray ionization mass spectrometry and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry in combination with selective enzymatic modifications in the characterization of glycosylation patterns in single-chain plasminogen activator

The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-link glycopeptides, which are present at low level ( < 1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.     

Purification and determination of intact molecular mass by electrospray ionization mass spectrometry of the photosystem II reaction center subunits

A reverse phase high pressure liquid chromatography purification system for the rapid separation of photosystem II reaction center proteins free of salts and detergents is described. This procedure results in the isolation of the three small subunits: alpha- and beta-subunits of cytochrome b559 and PsbI protein, with near base-line resolution between each peak, although the D1 and D2 proteins were partially deconvoluted. The molecular masses obtained by electrospray ionization mass spectrometry for the purified beta-subunit of cytochrome b559, alpha-subunit of cytochrome b559, and the PsbI protein, 4,394.8 +/- 0.4, 9,283.7 +/- 0.8, and 4,209.5 +/- 0.4 Da, respectively, are in excellent agreement with values obtained from previous characterization studies (Sharma, J., Panico, M., Barber, J., and Morris, H. R. (1997) J. Biol. Chem. 272, 3935-3943). Direct electrospray analysis of the D1 and D2 proteins suggests that these components exist in heterogeneous forms. The molecular mass ascribed to a predominant form of the D1 protein, 38, 040.9 +/- 6.5 Da, and the D2 protein, 39,456.1 +/- 7.7, are also in agreement with those expected for the mature nonphosphorylated states of these subunits.     

High-performance liquid chromatography-mass spectrometry for the quantification of nortriptyline and 10-hydroxynortriptyline in plasma

A highly sensitive and selective method for the quantification of nortriptyline and its major 10-hydroxy metabolite in plasma is described. The method is based on liquid-liquid extraction in combination with acid dehydration of the 10-hydroxy metabolite to the less polar 10,11-dehydronortriptyline. Deuterium labelled internal standards ([2H4]NT and [2H3]10-OH-NT) were used and the compounds were separated by reversed-phase HPLC and detected using atmospheric pressure chemical ionisation and mass spectrometry. The limit of quantification was 0.8 ng/ml for both compounds. A 1-ml volume of plasma was used for analysis in the concentration range 0.8-32 ng/ml. The within- and between-day coefficients of variation were 11% in the low, 1.6 ng/ml range, and 7% at 8 ng ml/ml. Using this method it was possible to quantify plasma concentrations for 168 h following a single oral dose of 25 mg of nortriptyline with good accuracy and precision.     

Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation

A new method based on protein fragmentation and directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC-FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2-3, 0 degrees C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC-FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h-1) to very slow (k < 0.002 h-1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC-FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC-FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.     

Defluorinated sparfloxacin as a new photoproduct identified by liquid chromatography coupled with UV detection and tandem mass spectrometry

Photodegradation of sparfloxacin was observed by means of high-pressure liquid chromatography with UV detection and liquid chromatography coupled with UV detection and tandem mass spectrometry (LC-MS/MS). Three products were detected. Comparison with an independently synthesized derivative of sparfloxacin revealed the structure of one product which is believed to be 8-desfluorosparfloxacin. The second product is likely to be formed by the splitting off of a fluorine and a cyclopropyl ring. Thus, photodefluorination of quinolone antibacterial agents is found and proved for the first time by LC-MS/MS.     

A microbore high performance liquid chromatography/electrospray ionization mass spectrometry method for the determination of the phytoestrogens genistein and daidzein in comminuted baby foods and soya flour

Motor vehicle crashes are a leading cause of death and disability among children in the United States despite the fact that child safety seats and seat belts can provide effective protection against serious and fatal injuries. Many child car seats and safety restraints are being used incorrectly. In addition, recent fatalities associated with children and air bags have heightened awareness of child passenger safety issues. Pediatric nurse practitioners are in an excellent position to offer motor vehicle occupant prevention strategies to families through health education and anticipatory guidance.