Ultra-high flow rate capillary liquid chromatography with mass spectrometric detection for the direct analysis of pharmaceuticals in plasma at sub-nanogram per millilitre concentrations.

Ultra-high flow rate liquid chromatography on large particle size stationary phases coupled with mass spectrometric detection (particularly tandem mass spectrometry, MS/MS) is gaining increasing usage for the direct determination of pharmaceuticals in biological fluids. The lack of sample preparation required prior to chromatographic and MS/MS analysis, together with the extremely high throughput of the chromatography, make the technique extremely attractive to the modern pharmaceutical bioanalyst. However, this lack of sample preparation also means that there is no potential for concentration of the sample and, as a consequence, the sensitivity of the technique has been limited. Liquid chromatography on the capillary scale offers sensitivity benefits compared with conventional liquid chromatography as the volume in which the analyte peaks are eluted is greatly reduced. In this paper, we present the use of ultra-high flow rate liquid chromatography on the capillary scale. This enables the quantification of drugs in plasma at sub-nanogram per millilitre concentrations from a very small (2.5 micromgL) aliquot of plasma without sample preparation. We also compare the resolution obtained by ultra-high flow rate liquid chromatography with that achieved on short columns packed with conventional size packing materials operated in an isocratic manner.

Characterization of immune response to Eimeria tenella antigens in a natural immunity model with hosts which differ serologically at the B locus of the major histocompatibility complex.

A model to simulate natural immunity to Eimeria tenella was developed in three chicken lines which differ at the B locus of the major histocompatibility complex. Homozygous, 1-day-old chicks of the B19B19, B24B24, or B30B30 genotype were trickle immunized by being orally fed a small infectious dose of E. tenella oocysts for 5 consecutive days. These naturally exposed birds were then challenged at different times between 5 and 24 days after the final dose, and the level of protection was assessed 6 days after challenge, using body weight gain and intestinal lesion scores. The duration of immunity in naturally exposed birds differed among the major histocompatibility complex lines. Trickle immunization of the B19B19 haplotype afforded the longest and strongest level of protection compared to the other two haplotypes tested. In addition, in vitro splenic and peripheral blood lymphocyte proliferative responses in trickle-immunized birds were measured against sporozoite, merozoite, and tissue culture-derived E. tenella parasite antigens isolated from the recently described SB-CEV-1/F7 established cell line. The lymphocytes obtained from B19B19 birds trickle immunized responded in vitro to the E. tenella-infected SB-CEV-1/F7 tissue culture-derived parasite antigen. Furthermore, antigen-specific immune responses appeared earlier in immune, challenged B19B19 birds than in their naive, challenged counterparts. The development of a model simulating natural immunization will serve as a foundation to further characterize both humoral and cell-mediated responses to E. tenella tissue culture-derived parasite antigens and to better understand host protective immune responses to avian coccidiosis.

Determination of the acylcoenzyme A cholesterol acyltransferase inhibitor 447C88 in plasma using gas chromatography-mass spectrometry and liquid chromatography atmospheric pressure chemical ionisation tandem mass spectrometry.

Two mass spectrometry-based methods are described for the determination of 447C88 (I), a novel inhibitor of acylcoenzyme A cholesterol acyltransferase (ACAT), in rat, dog and human plasma. The first method uses gas chromatography-mass spectrometry (GC-MS) with electron ionisation and selected-ion monitoring. The method employs solid-phase extraction of I from plasma and requires alkylation of I using iodoethane. The second method uses liquid chromatography-tandem mass spectrometry (LC-MS-MS) with atmospheric-pressure chemical-ionisation and selected-reaction monitoring. The LC-MS-MS method uses a simplified version of the extraction procedure used for GC-MS and does not require derivatisation of I. While both methods provide the necessary limit of quantitation of 0.5 ng/ml in human, dog and rat plasma with the required precision and accuracy, the LC-MS-MS assay offers increased sensitivity, selectivity and speed over the GC-MS assay. This allows a same day turn round of results for in excess of 100 samples, including sample preparation and data acquisition and processing.

Ultrastructural observations of development of Eimeria tenella in a novel established avian-derived cell line.

Transmission electron microscopy was used to study the in vitro development of Eimeria tenella in a novel established avian-derived cell line (designated CEV-1/F7) used for antigen production in chicken immunization studies. Sporozoites of E. tenella were inoculated onto cell monolayers and the cells were fixed at 24-h intervals. Large numbers of intracellular sporozoites were seen at 24 h postinoculation (p.i.), and trophozoites were identified at 24-48 h p.i. Immature schizonts, some with budding merozoites, were seen by 48 h p.i. At 72-96 h p.i., immature and mature schizonts and extracellular merozoites were observed. No merozoite invasion occurred, but immature second-generation schizogony was seen in parasitophorous vacuoles of first-generation schizonts. No further development occurred and degeneration of most schizonts was seen by 120-144 h p.i. The results confirmed synchronous development of E. tenella until 48 h p.i., followed by asynchronous development and ultrastructural degeneration with increased incubation time.

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 beta-glucuronidase preparation from Helix pomatia.

Gas chromatographic-mass spectrometric characterisation of some novel hydroxyeicosatetraenoic acids formed on incubation of arachidonic acid with microsomes from induced rat livers.

The biotransformation of arachidonic acid by rat liver microsomes from both control animals and animals pretreated with known inducers of cytochrome P-450 isoenzymes has been studied using a combination of reversed- and normal-phase high-performance liquid chromatography and combined gas chromatography-electron-impact mass spectrometry. The metabolite profiles observed were found to be dependent upon the inducing agent. Five metabolites were identified, namely 16-, 17-, 18-, 19- and 20-hydroxylated arachidonic acids. Of these the 16- and 17-isomers have not been reported as products of arachidonic acid metabolism by any biological system and the 18-isomer has not been reported as a product of liver metabolism.

Characterization of resistance and immunity to Eimeria tenella among major histocompatibility complex B-F/B-G recombinant hosts.

The relative influence of the B-F vs. B-G chromosomal regions on innate resistance and immunity to Eimeria tenella was studied among six B-F/B-G recombinants, designated BR1, BR3, BR4, BR5, BR6, and BR8. In one experiment, resistance was studied among 340 F1 chicks each carrying the B17 allele derived from Line UCD.003 and therefore also heterozygous for one of the B recombinant haplotypes. In a second experiment, acquired immunity was studied among 161 F2 chicks each carrying one B17 allele and segregating for one of the recombinant alleles. In Experiment 1, F1 chicks of similar haplotypes, BR3 and BR4 (BF2-G23), gained significantly more weight than those with BR1 (BF24-G23) and BR5 (BF21-G19) following exposure to a single high dose of 25,000 oocysts, although no differences in lesion scores were detected among the six recombinants. Following exposure to a single low dose of 2,500 oocysts, F1 chicks BR3 and BR4 also gained significantly more weight and had significantly lower lesion scores than BR1 or BR5 F1 chicks. To study acquired immunity in the F2 homozygous chicks, five consecutive daily exposures of 500 oocysts were followed 21 days later by challenge with 10,000 oocysts. The BR5 (BF21-G19) and BR6 (BF21-G23) homozygous chicks, both carrying the B-F21 allele, showed significant protection in terms of reduced lesion scores. These results demonstrate that E. tenella parasitism manifests itself to varying degrees in chicken hosts possessing different recombinant major histocompatibility complex haplotypes.

Major histocompatibility complex control of immunity elicited by genetically engineered Eimeria tenella (Apicomplexa) antigen in chickens.

The immunogenicity of a recombinant Eimeria tenella coccidial antigen was studied in 6(1).B congenic chickens derived from B2B2 and B5B5 parents segregating for haplotypes B2 and B5. Five-week-old chickens were immunized with 2.4 micrograms of recombinant protein (designated 5401) in Freund complete adjuvant and challenged with 75,000 oocysts at 28 days postimmunization (DPI) to determine the degree of elicited protective immunity. Serum samples were collected weekly for 5 weeks postimmunization for analysis by enzyme-linked immunosorbent assay, immunofluorescence assay, and Western blotting. Lesion scores following oocyst challenge were significantly reduced in B5B5 chickens compared with those in B2B2 chickens. Immunization induced a sporozoite-specific immunoglobulin G (IgG) titer in serum detected by the enzyme-linked immunosorbent assay that peaked at 28 DPI, the day of challenge, in B5B5 chickens and at 42 DPI in B2B2 chickens. After challenge, this titer declined for each genotype. Anti-sporozoite IgG detected by the immunofluorescence assay attained a peak titer at 21 DPI in B2B2 chickens and 28 DPI in B5B5 chickens. Serum from immunized B5B5 chickens reacted strongly in Western blots with several high-molecular-weight (greater than 100,000), soluble proteins prepared from sporozoites. Serum from B2B2 chickens reacted with similar proteins as well as with a 51- to 53-kilodalton protein that was not labeled by serum from B5B5 chickens. These results demonstrate further the role of host genetics on anticoccidial immunity and suggest that a peak anti-sporozoite IgG titer in B5B5 chickens on the day of challenge may signal a state of immunocompetence to that challenge.

Chemical burns secondary to elemental metal exposure: two case reports.

Elemental metal may react when exposed to water or moist air, producing both chemical and thermal burns. Two cases are reviewed: one involving the outpatient treatment of burns secondary to an elemental sodium exposure, and one involving the inpatient treatment of burns secondary to an elemental potassium exposure. Water is contraindicated in the initial management of such exposures. The wound should be covered with oil until removal of any unreacted metal is completed. Burns may then be regarded similarly to alkali exposures.

Anti-Rous sarcoma response of major histocompatibility (B) complex haplotypes B23, B24 and B30.

Responses to Rous sarcoma virus (RSV) induced tumours were studied in UNH 105, a non-inbred line of New Hampshire chickens. Six single male matings encompassing a total of 50 dams produced 345 progeny which segregated for B complex genotypes B23/B23, B23/B24, B23/B30, B24/B24, B24/B30 and B30/B30. Six-week-old chicks were wingweb inoculated with a pseudotype of Bryan high titre Rous sarcoma virus, BH RSV (RAV-1). Tumours were scored for size six times over a 10-week period post-inoculation. Each chick was assigned a tumour profile index (TPI) as an indicator of immunological response. The number of days to death (DTD) was recorded for 148 chicks with terminal tumours. Genotypes B23/B23, B23/B24 and B23/B30, with TPIs of 1.8, 1.7 and 2.0 respectively, did not differ significantly from each other, suggesting dominance of response of B23 over B24 and B30 haplotypes. B24/B30 chicks with the highest TPI (3.4) and shortest DTD (34.6) were significantly different from B30/B30 (2.8; 41.6) but not from B24/B24 (3.1; 34.9) suggesting dominance of response of the B24 haplotype over B30 in the absence of B23.

Bile and serum immunoglobulin levels during primary and secondary infections with Eimeria tenella in chickens.

Using a radial immunodiffusion assay, total bile and serum IgG, IgM and IgA were measured following primary and secondary exposures to Eimeria tenella. Neither IgG nor IgM could be detected consistently in bile. Biliary IgA peaked at Days 6 and 10 following a primary infection of either 5000 or 10,000 oocysts and remained elevated following a subsequent 10,000-oocyst challenge at Day 10. Serum IgG and IgM levels were not influenced by parasitism and measurable concentrations of serum IgA were not detected.

Delayed wattle reactions in Eimeria tenella infected chickens.

Wattle reactions to an Eimeria tenella antigen and phytohemagglutinin (PHA) were studied in chickens infected with E. tenella. Two trials were conducted using a total of 224 chickens. Four days after infection with one dose of 10,000 sporulated oocysts, the increase in wattle thickness in response to E. tenella antigen was significantly greater than that of uninfected controls. This significant response persisted through day 10 post infection. Wattle response to PHA 3 days after infection were significantly greater than for uninfected controls. Significant differences in response to PHA were maintained throughout the experiment except on day 6. An increased response to PHA from days 7 to 13 post infection was attributed to the lower parasite burden at that time.

Plasma hydrazine concentrations in man after isoniazid and hydralazine administration.

By using a specific sensitive stable-isotope dilution gas chromatography-mass spectrometry (GC-MS) assay, hydrazine was detected in the plasma of eight healthy male volunteer subjects taking isoniazid (300 mg daily) for 2 weeks. Accumulation of hydrazine occurred in slow-acetylator phenotypes. Hydrazine was also detected in the plasma of eight out of 14 hypertensive patients treated chronically with hydralazine (200 mg daily). However, the concentrations of hydrazine observed were much lower than in the isoniazid study and were close to the limit of detection. As hydrazine is hepatotoxic, mutagenic and carcinogenic in animals, its presence in human plasma has important toxicological implications.

Major histocompatibility (B) complex effects on acquired immunity to cecal coccidiosis.

The influence of the major histocompatibility (B) complex on acquired immunity to the avian coccidium Eimeria tenella was studied in 217 F4 segregants (B2B2, B2B5, B5B5) of a cross between inbred lines 6(1) (B2B2) and 15(1) (B5B5) and segregating haplotype combinations of UNH105 (B23B23, B23B24, B24B24), a noninbred line of New Hampshire chickens. Chickens were immunized at 6 weeks of age with 500 oocysts daily for 5 days, then challenged 14 days later with 10 000 oocysts. Responses to infection were evaluated by cecal lesion scores, body weight gain, delayed wattle reaction (DWR), and spleen weight. The F4 segregants of genotypes B2B5 and B5B5 exhibited greater immunity to challenge than B2B2 chickens. B5B5 chickens showed a significantly greater DWR following immunization and larger spleens 6 days after the challenge than either of the other genotypes. However, both B2B5 and B5B5 chickens demonstrated significantly lower lesion scores than B2B2 chickens. There were no significant differences in weight gain among these genotypes. Among 139 line UNH105 segregants, B23B23 hosts had significantly lower lesion scores than B24B24 chickens. No other differences in immune response among line UNH105 genotypes were detected.

Metabolism of hydroxy sterols by rat liver.

5 alpha-[16-3H]lanost-8-ene-3 beta,15 alpha-diol and 5 alpha-[16-3H]lanost-8-ene-3 beta,15 beta-diol were both extensively metabolised by rat liver enzymes in vitro. Quantitatively, the most important product in both cases was a more polar compound, tentatively identified as a 5 alpha-lanost-8-enetriol. In addition, 5 alpha-[16-3H]lanost-8-ene-3 beta,15 beta-diol gave rise to the corresponding 3 beta,15beta-diol diester, whilst with 5 alpha-[16-3H]lanost-8-ene-3 beta,15 alpha-diol only the 3 beta-hydroxyl group was esterified. The enzymes involved may normally be responsible for metabolising spontaneously produced non-enzymic oxidation products of dietary or cellular cholesterol. High concentrations of 5 alpha-[16-3H]lanost-8-ene-3 beta,15 beta-diol stimulated ester formation. With both substrates, carbon monoxide inhibited formation of the polar sterol metabolite but stimulated ester formation. Under all conditions, cholesterol was a relatively minor metabolic product of either of the 5 alpha-lanost-8-ene-3 beta,15-diols.

The analysis of terbutaline in biological fluids by gas chromatography electron impace mass spectrometry.

A highly sensitive and specific assay is described for the bronchodilator drug terbutaline in human plasma and urine, based on single ion monitoring gas chromatography mass spectrometry and employing a homologue of the drug as internal standard. Recovery of terbutaline and internal standard into ethyl acetate is effected at pH 9.8, while back-extraction into dilute acid serves to purify the initial extract. Following preparation of O-TMS, N-TFA derivatives, the drug and its homologue are detected by selected ion monitoring of their common base ion at m/e 355. The limit of detection of terbutaline by this procedure is 0.3 ng ml-1 from a 4 ml sample of plasma.

Quantitative determination of the herbicide paraquat in human plasma by gas chromatographic and mass spectrometric methods.

The gas chromatographic (GC) determination of the herbicide paraquat, the 1,1'-dimethyl-4,4'-dipyridyl cation in human plasma is described. In poisoning cases, plasma concentrations provide a necessary index of the severity of intoxication and a means of monitoring subsequent therapy. The methods may be extended to the specific trace analysis of paraquat in body fluids of post-mortem tissue. Reduction of fully ionised paraquat salts with sodium borohydride yields a hexahydro derivative, a diene, amenable to solvent extraction and GC. Employing 1,1'-diethyl-4,4'-dipyridyl dichloride as the internal standard, plasma concentrations of 0.1 microgram/ml (+/- 6% S.D.) may be determined with flame ionisation detection and 0.025 microgram/ml with nitrogen-selective flame ionisation. Further enhancement of specificity is achieved using selected ion monitoring mass spectrometry and the value of this technique in forensic analysis is illustrated.

Maternal and neonatal elimination of amobarbital after treatment of the mother with barbiturates during late pregnancy.

Plasma half-lives of amobarbital were determined in newborn children of 10 mothers who had been treated with barbiturates for hypertension in pregnancy for 6 to 42 days prior to delivery. Five mothers had received amobarbital, 200 mg daily, and 5, phenobarbital, 60 to 180 mg daily. Half-lives in 7 of the babies ranged from 16.6 to 49.4 hr, comparable to those previously reported in babies of mothers who had received only a single dose of amobarbital. Thus there was no evidence of induction of amobarbital hydroxylation in these children. Two babies who had a greater than normal rise in serum bilirubin had longer half-lives (86.1 and 117.7 hr). In 1 baby whose mother had membranous glomerulonephritis, plasma amobarbital concentration did not significantly change over the period of the study.