Immunoaffinity column cleanup procedure for analysis of ivermectin in swine liver

A simple and sensitive method for the analysis of ivermectin (22,23-dihydroavermectin B₁) in swine liver based on immunoaffinity column cleanup is described. The immunosorbent was prepared by coupling polyclonal anti-ivermectin antibodies to carbonyl diimidazole-activated Sepharose CL-4B. After extraction with methanol, ivermectin was cleaned up on an immunoaffinity column, and determined by reversed-phase liquid chromatography with UV absorbance detection at 245 nm. Recoveries of ivermectin from fortified samples of 5–100 μg kg⁻¹ levels ranged 85–102%, with coefficients of variation of 6–12%. The limit of detection was 2 μg kg⁻¹ in a 5-g sample.     

Fluorescent derivatives of prostaglandins and thromboxanes for liquid chromatography

Fluorescent esters of the prostablandins D₂, E₂, F₂α, and 6-keto-F₁α and of thromboxane B₂ have been prepared using the reagent 4-bromomethyl-7-methoxycoumarin. All of these derivatives can be separated in a single run either by thin-layer or high-performance liquid chromatography (TLC or HPLC). As little as 20 ng of PGE₂ can be detected after derivatization and HPLC analysis. Identification of thromboxane B₂ produced by human platelets and of 6-keto-PG F₁α produced by bovine aortic microsomes has been achieved with this method.     

Determination of the GABAB receptor agonist CGP 44532 (3-amino-2-hydroxypropylmethylphosphinic acid) in rat plasma after pre-column derivatization by micro-high-performance liquid chromatography combined with negative electrospray tandem mass spectrometry

An assay, based on pre-column derivatization and micro-high-performance liquid chromatography–tandem mass spectrometry, was developed for the determination of the GABA_B agonist CGP 44532 in rat plasma. CGP 44532, a highly polar 3-amino-2(S)-hydroxypropylmethylphosphinic acid, presented difficulties in developing a chromatographic method for the analysis of the compound in rat plasma. Instead of analyzing the target compound directly, it was derivatized prior to separation to a 4-nitrobenzylcarbamate isopropyliden derivative. In order to reach the required quantitation limit, on-line solid-phase extraction was utilized for sample clean-up and reversed-phase micro-column high-performance liquid chromatography, for separation of the plasma samples. The separated compounds were detected by negative electrospray tandem mass spectrometry in selected reaction monitoring mode. The derivatives show good chromatographic and mass spectrometric properties and both the target compound and the internal standard, could be eluted as symmetrical peaks with good signal/noise ratio. The MS–MS detection was selective and sensitive due to the straight fragmentation pattern. After injection of 200-μl sample aliquots, the limit of quantification was 10 ng ml⁻¹. The analytical assay is useable in the range of 10–500 ng ml⁻¹.     

Biosynthesis, characterization and inhibition of leukotriene B4 in human whole blood

We have evaluated the biosynthesis, characterization and inhibition of Leukotrien (LT) B₄ in unstimulated and in A23187-stimulated human whole blood. LTB₄ was assayed by radioimmunoassay (RIA) both in unextracted serum and after extraction and thin-layer chromatography (TLC). Unstimulated human whole blood allowed to clot at 37°C for 60 min produced only trace amounts of LTB₄ (0.16±0.05 ng/ml, mean±SD, n=3). LTB₄-like immunoreactivity (ir-LTB₄) detectable in unstimulated serum samples was largely overestimated by direct RIA, most likely because of interfering substance(s) unrealed to cyclooxygenasep or lipoxygenase activity. Incubation of human whole blood with A23187 (2–10 μM) resulted in a concentration-dependent stimulation of LTB₄ production. At 10 μM A23187, ir-LTB₄ was 18±2.4 ng/ml (mean±SEM, n=28). In A23187-stimulated serum samples, LTB₄ concentrations measured by direct RIA correlated in a statistically significant fashion with those measured after extraction and TLC. Nafazatrom added caused a dose-dependent inhibition of A23187-stimulated ir-LTB₄ production with an IC₅₀ of 17 μM.     

Development of a competitive enzyme immunoassay for 17α-19-nortestosterone

Because 17β-19-nortestosterone and its esters are frequently used anabolic steroids in cattle in Europe, there is a need for an assay that can also detect certain metabolites. The enzyme immunoassay described here involves the detection and quantitation of the major metabolite 17α-19-nortestosterone in urine. The assay is based on the coating of an antibody raised in a rabbit against 17α-19-nortestosterone-3-carboxy-methyloxime—bovine serum albumin (17α-19-NT-3-CMO-BSA), the competitive incubation of 17α-19-NT and the 17α-19-nortestosterone-3-CMO—horseradish peroxidase label, followed by the detection of the blue colour developed by the action of the enzyme on tetramethylbenzidine. The 3-CMO conjugate of 17α-19-nortestosterone was used to produce an antibody with selective affinity for the 17α-epimer. For the optimization of the assay, different coatings and incubation conditions were tested. The standard curve ranged between 0.98 and 4000 pg per well, with a B/B₀ 50% of ± 65 pg per well. Colour was measured with a microtitre plate reader. The method was validated by means of certified blank and spiked cattle urine samples.     

Ear-rot fungi and mycotoxins in South African corn of the 1989 crop exported to Taiwan

A shipment of South African corn (1989) exported to Taiwan, was analyzed for various ear-rot fungi andFusarium mycotoxins. Two sets of samples, one from the points of origin in South Africa prior to shipment, and the other from the end-point distributors in Taiwan, were studied. Surface-sterilized kernels were plated onto two different agar media and the fungal colonies identified. High Performance Liquid Chromatography was used to analyze mycotoxin levels. The predominant ear-rot fungi, in decreasing order of isolation frequency, wereFusarium subglutinans, F. moniliforme, Diplodia maydis andF. graminearum. Aspergillus flavus andA. parasiticus were not isolated from samples prior to export, but a small number ofA. flavus isolates were found after shipment. The predominant mycotoxins were fumonisins B₁ (0–865 ng/g) and B₂ (0–250 ng/g). Low levels of moniliformin (390 ng/g) were detected in some samples before shipment. Zearalenone (25 ng/g), and nivalenol (120 ng/g) were detected in two out of 32 samples taken in Taiwan. The samples contained no detectable levels of either aflatoxins (>0.5 ng/g) or deoxynivalenol (>100 ng/g) before or after shipment.Abbreviations RSA South Africa(n) - FB₁ fumonisin B₁ - FB₂ fumonisin B₂ - ETVL eastern Transvaal - WTVL western Transvaal     

Simultaneous determination of four anthracyclines and three metabolites in human serum by liquid chromatography–electrospray mass spectrometry

A sensitive and very specific method, using liquid chromatography–electrospray mass spectrometry (LC–ES-MS), was developed for the determination of epirubicin, doxorubicin, daunorubicin, idarubicin and the respective active metabolites of the last three, namely doxorubicinol, daunorubicinol and idarubicinol in human serum, using aclarubicin as internal standard. Once thawed, 0.5-ml serum samples underwent an automated solid-phase extraction, using C₁₈ Bond Elut cartridges (Varian) and a Zymark Rapid-Trace robot. After elution of the compounds with chloroform–2-propanol (4:1, v/v) and evaporation, the residue was reconstituted with a mixture of 5 mM ammonium formate buffer (pH 4.5)–acetonitrile (60:40, v/v). The chromatographic separation was performed using a Symmetry C₁₈, 3.5 μm (150×1 mm I.D.) reversed-phase column, and a mixture of 5 mM ammonium formate buffer (pH 3)–acetonitrile (70:30, v/v) as mobile phase, delivered at 50 μl/min. The compounds were detected in the selected ion monitoring mode using, as quantitation ions, m/z 291 for idarubicin and idarubicinol, m/z 321 for daunorubicin and daunorubicinol, m/z 361 for epirubicin and doxorubicin, m/z 363 for doxorubicinol and m/z 812 for aclarubicin (I.S.). Extraction recovery was between 71 and 105% depending on compounds and concentration. The limit of detection was 0.5 ng/ml for daunorubicin and idarubicinol, 1 ng/ml for doxorubicin, epirubicin and idarubicin, 2 ng/ml for daunorubicinol and 2.5 ng/ml for doxorubicinol. The limit of quantitation (LOQ) was 2.5 ng/ml for doxorubicin, epirubicin and daunorubicinol, and 5 ng/ml for daunorubicin, idarubicin, doxorubicinol and idarubicinol. Linearity was verified from these LOQs up to 2000 ng/ml for the parent drugs (r≥0.992) and 200 ng/ml for the active metabolites (r≥0.985). Above LOQ, the within-day and between-day precision relative standard deviation values were all less than 15%. This assay was applied successfully to the analysis of human serum samples collected in patients administered doxorubicin or daunorubicin intravenously. This method is rapid, reliable, allows an easy sample preparation owing to the automated extraction and a high selectivity owing to MS detection.     

The production of cyclopiazonic acid by Penicillium commune and cyclopiazonic acid and aflatoxins by Aspergillus flavus as affected by water activity and temperature on maize grains

The combined effects of water activity (a_w) and temperature on mycotoxin production by Penicilium commune (cyclopiazonic acid — CPA) and Aspergillus flavus (CPA and aflatoxins — AF) were studied on maize over a 14-day period using a statistical experimental design. Analysis of variance showed a highly significant interaction (P 0.001) between these factors and mycotoxin production. The minimum a_w/temperature for CPA production (2264 ng g^–1 P. commune, 709 ng g^–1 A. flavus) was 0.90 a_w/30 °C while greatest production (7678 ng g^–1 P. commune, 1876 ng g^–1 A. flavus) was produced at 0.98 a_w/20 °C. Least AF (411 ng g^–1) was produced at 0.90 a_w/20 °C and most (3096 ng g^–1) at 0.98 a_w/30 °C.     

Population growth rates forAmblyomma americanum (Acari: Ixodidae) onBos indicus,B. taurus andB. indicus x B. taurus cattle

Densities ofAmblyomma americanum (L.) onBos indicus, B. taurus andB. indicus x B. taurus cattle are compared over a 3-year period, and the growth rate (rate of increase or decrease) of parasitic tick populations on each cattle genotype is estimated.Average log₁₀ densities of parasiticA. americanum larvae are significantly (P=0.05) lower onB. indicus cattle than onB. taurus andB. indicus x B. taurus cattle. Average log densities of nymphal and adult ticks onB. taurus cattle are significantly higher than onB. indicus cattle but neither cattle genotype differs in this regard fromB. indicus x B. taurus cattle.Estimated annual tick population growth rates (log₁₀) for parasiticA. americanum are positive onB. taurus cattle (+0.84 larvae, +0.09 nymphs, +0.22 adults calf^–1 year^–1), but are negative onB. indicus (–0.18 nymphs, –0.14 adults calf^–1 year^–1) andB. indicus x B. taurus cattle (–0.45 larvae, –0.24 nymphs, –0.14 adults calf^–1 year^–1). Populations of parasitic larvae were not detected onB. indicus cattle.     

Determination of the tricyclic compound adosupine and its three metabolites in plasma and brain of rat using high-performance liquid chromatography

An analytical method for the detection in biological samples of the novel tricyclic compound adosupine (10-acetoamido-5-methyl-5,6-dihydro-11H-dibenzo[b,e]azepin-6,11-dione), which is capable of influencing various forms of urinary bladder hyperreflexia has been developed using high-performance liquid chromatography with UV detection. Liquid—liquid extraction was used to isolate the parent compound, three metabolites and an analogue (added as internal standard) from plasma and brain of rat. Adosupine was well separated from its three metabolites with 0.01 M disodium hydrogenphosphate—acetonitrile—methanol—nonylamine (59.986:38:2:0.014) at pH 4.5 as mobile phase using a C₁₈ reversed-phase column. The standard curves were linear in the range 50–5000 ng/ml (or ng/g) for adosupine and metabolites in both plasma and brain. The between- and within-assay variations for high and low concentrations of the parent compound and the three metabolites were 8.2–14%. In the range 50–5000 ng/ml (or ng/g) the accuracy of the method was satisfactory, with the relative error always lower than 10%. Analytical recoveries of added adosupine and the three metabolites were higher than 82%. The method has been applied successfully, to investigate the pharmacokinetics of the drug and its distribution in the central nervous system of rats.     

Determination of urinary glucuronide conjugates by high-performance liquid chromatography with pre-column fluorescence derivatization

A simple and highly sensitive high-performance liquid chromatographic method for the direct determination of urinary glucuronide conjugates is described. The method is based on the direct derivatization of the glucuronic acid moiety in glucuronide conjugates with 6,7-dimethoxy-1-methyl-2 (1 H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 0–37°C. The resulting fluorescent derivatives are separated on a C₁₈ column using methanol—acetonitrile—0.5% triethylamine in water (1:1:2, v/v) as mobile phase, and are detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise RATIO = 3) for the glucuronides are 13–48 fmol for an injection volume of 10 μl (130–480 fmol per 5 μl of human urine). The method was applied to the measurement of etiocholanorone-3-glucuronide and androsterone-3-glucuronide in human urine. The method is simple and rapid without conventional liquid—liquid extraction of the glucuronides from urine.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

o-Phthalaldehyde–N-acetylcysteine polyamine derivatives: formation and stability in solution and in C18 supports

A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10⁻² min⁻¹ in solution versus 7.7±1.1×10⁻⁴ min⁻¹ on the (C₁₈) solid support. The results obtained showed that forming the derivative on the packing support (C₁₈) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples.     

Electron capture gas chromatographic detection of thromboxane B2

An electron capture gas chromatographic method is described for the detection of thromboxane B₂. Thromboxane B₂ is esterified with diazomethane, followed by treatment with pentafluorobenzylhydroxylamine hydrochloride and silylation with BSA. In pyridine, the free aldehyde form of the acetal ring is favored allowing rapid formation of a novel thromboxane B₂ pentafluorobenzyloxime. The method has been applied to detect thromboxane B₂ formation during aggregation of washed platelets. It must be emphasized that by ordinary analytical standards, the derivatization reproducibility from 50–375 nanograms is poor (±11% – ±42%); however, the improved selectivity of the method and its ability to detect nanogram levels of thromboxane B₂ make it a useful complement to commonly employed bioassay techniques.     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

Control of induced infestations of three African multihost tick species with sustained-release ivermectin

The efficacy of ivermectin, released intraruminally from a 28-day-delivery device was evaluated in two titration studies against induced infestations of adultRhipicephalus appendiculatus, R. evertsi andHyalomma truncatum on cattle. Cattle were given a sufficient number of devices to release ivermectin at approximately 20, 40, 60 or 80 g kg^–1 day^–1 at a steady-state rate 7–28 days after administration. Tick mortality was recorded, engorged female ticks were weighed and individually incubated, and reproductive data were recorded to determine a reproductive index for the species at various dose levels. Mortality of male and female ticks compared to that of controls was directly related to the daily dose of ivermectin, as was the number of ticks not engorging. Ticks fed on ivermectin-treated cattle had a smaller mass when engorged and laid smaller egg-masses, both absolutely and as a proportion of engorged mass.The index of reproduction ofR. appendiculatus was reduced by more than 99.9% at 20 g kg^–1 day^–1, and the reproductive indices ofR. evertsi andH. truncatum were reduced by more than 99.9% at dose rates of 40 g kg^–1 day^–1 and above.Practical implications of the application of sustained-release ivermectin for the control of multihost ticks and tick-borne diseases are discussed.     

Determination of seven prostanoids in 1 ml of urine by gas chromatography—negative ion chemical ionization triple stage quadrupole mass spectrometry

In an isotope dilution assay, prostaglandin (PG) E₂, 6-keto-PGF_1α, thromboxane (Tx) B₂ and their metabolites PGE-M (11α-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid), 2,3-dinor-6-keto-PGF_1α, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂ were determined in urine by gas chromatography—triple stage quadrupole mass spectrometry (GC—MS—MS). After addition of deuterated internal standards, the prostaglandins were derivatized to their methoximes and extracted with ethyl acetate—hexane. The sample was further derivatized to the pentafluorobenzylesters and purified by thin-layer chromatography (TLC). Three zones were scraped from the TLC plate. The prostanoid derivatives were converted to their trimethylsilyl ethers and the products were quantified by GC—MS—MS. In each run, two or three prostanoids were determined.     

The effect of avermectins on feeding,salivary fluid secretion,and fecundity in some ixodid ticks

We tested the effects of the potent acaricides, avermectin B₁a (AVM) and 22,23-dihydroavermectin B₁ (ivermectin; IVM) when injected directly into partially fed and fully engorged female ticks. When injected into small ticks (Amblyomma hebraeum Koch), neither drug (up to 100 g/kg b.w.) inhibited subsequent engorgement nor affected oviposition latency, weight of total egg mass laid nor viability of laid eggs. At higher concentrations (1000 and 5000 g/kg b.w.), AVM and IVM were markedly toxic. When injected into engorged ticks, both drugs increased oviposition latency, and reduced fecundity at about 75–100 g/kg b.w. Vitellogenesis, as assessed by a spectrophotometric assay of the ovaries, was not inhibited. Also at 50–100 g/kg b.w., AVM and IVM caused paralysis of the abdominal dorso-ventral muscles and the leg muscles. Both drugs, at 7 days post-injection, proved detrimental to salivary gland function in both small and large ticks, but had little effect on salivary gland weight. At concentrations which did not inhibit oviposition (20–50 g/kg b.w.) many of the eggs dried out even though they were kept at high RH. We then demonstrated inAmblyomma americanum, Dermacentor andersoni andD. albipictus that removal of egg wax (by extraction with hexane) induced a marked increase in water permeability. IVM neither increased water permeability ofD. andersoni eggs nor diminished the amount of egg wax deposited on the surface of the eggs, when injected posteriorly through the alloscutum. However, injection of IVM, dimethylsulphoxide (vehicle for IVM) or distilled water through the articulation between the capitulum and scutum (anterior injection), did markedly reduce the wax coating and increased egg permeability. We suggest that anterior injection damages Gené's organ and thus causes the latter effects.     

In-feed 0.6% ivermectin formulation for treatment of wild boar in the Moslavina hunting ground in Croatia

An in-feed 0.6% ivermectin formulation was administered for 7 days to wild boar piglets at three sites of the Moslavina hunting ground in Croatia. Examination of internal organs and skin of five piglets that died immediately before the start of administration of the ivermectin formulation revealed the presence of Metastrongylus apri and Metastrongylus pudendotectus in the lungs, and of Ascarops strongylina, Physocephalus sexalatus and Globocephalus urosubulatusin in the gastrointestinal tract. Coccidial oocysts were found in the feces of all animals. Sarcoptes scabiei var. suis was identified in the skin of four piglets. The efficacy of treatment was assessed by examining fecal samples before start of therapy (day 0) and on days 7 and 14. Before treatment strongylid-type eggs were detected in 70–100% of fecal samples (210–505 EpG) The eggs of Strongyloides ransomi, Trichuris suis, Ascaris suum, Ascarops strongylina and Physocephalus sexalatus were identified in 10–50% of fecal samples at an intensity of 5–45 EpG. On day 14 after the start of the treatment, strongylid-type eggs were detected in 10% of fecal samples from one of the three sites only. Eggs of other helminth species were not detected at any of the three sites. This confirmed the successful therapeutic efficacy of the in-feed 0,6% ivermectin formulation.     

Determination of temazepam and temazepam glucuronide by reversed-phase high-performance liquid chromatography

A rapid and sensitive method for extracting temazepam from human serum and urine is presented. Free temazepam is extracted from plasma and urine samples using n-butyl chloride with nitrazepam as the internal standard. Temazepam glucuronide is analyzed as free temazepam after incubating extracts with β-glucuronidase. Separation is achieved using a C₈ reversed-phase column with a methanol—water—phosphate buffer mobile phase. An ultraviolet detector operated at 230 nm is used and a linear response is observed from 20 ng/ml to 10 μg/ml. The limit of detection is 15.5 ng/ml and the limit of quantitation is 46.5 ng/ml. Coefficients of variation are less than 10% for concentrations greater than 50 ng/ml. Application of the methodology is demonstrated in a pharmacokinetic study using eight healthy male subjects.