Weak Cation-exchange Solid-phase Research Articles

Determination of bencycloquidium bromide in human urine using weak cation-exchange solid-phase extraction and LC–ESI-MS: Method validation and application to kinetic study of urinary excretion

A sensitive LC–ESI-MS method has been developed and validated for the determination of bencycloquidium bromide (BCQB) in human urine samples. The method utilized a solid-phase extraction (SPE) procedure, choosing carboxy propyl phase (CBA) as the extracting sorbent for purification of BCQB, with better baseline and higher selectivity achieved. Sample preparation by this method yielded very good and consistent mean recovery of above 94.5%. Another major benefit of the present method was the high detectability, with a lower limit of quantification (LLOQ) of 0.02 ng/ml. The developed method was successfully applied to determine BCQB in human urine, and was proved to be suitable for use in Phase I clinical pharmacokinetic study of BCQB.

Solid phase extraction and liquid chromatography–electrospray ionization–mass spectrometry for the determination of bencycloquidium bromide in human plasma

To support the clinical pharmacokinetic trials of bencycloquidium bromide (BCQB), a specific and sensitive method based on weak cation-exchange solid phase extraction (WCX-SPE) and HPLC–ESI–MS techniques for the determination of BCQB in human plasma was developed and validated. BCQB and the internal standard 1-ethyl-bencycloquidium bromide were separated on a Zorbax Eclipse Plus C18 column (3.5 μm, 150 mm × 2.1 mm i.d.) with a mobile phase consisted of 20 mM ammonium acetate buffer solution containing 1% acetic acid (pH 3.6)–methanol (50:50, v/v), and the chromatographic run time for one sample was 8 min. The lower limit of quantitation (LLOQ) of the method is 5 pg/ml, which is critically important for the pharmacokinetic study of BCQB. The method possesses a reliable quantification range of 5–1000 pg/ml, the acceptable intra- and inter-batch precision of less than 8.9%, and the extraction recovery of more than 90.6%. The method was successfully applied for the single-dose pharmacokinetic study of BCQB nasal spray in healthy Chinese volunteers.

A generic method for the quantitative analysis of aminoglycosides (and spectinomycin) in animal tissue using methylated internal standards and liquid chromatography tandem mass spectrometry

Aminoglycosides (AGs) are a large and diverse group of antibiotics. Although AGs may cause side effects of nephrotoxicity and ototoxicity, they are still occasionally being used for the treatment of serious infections. In this study the development of a method is described for the quantitative determination and confirmation of seven aminoglycosides (and relevant isomers) and spectinomycin in animal tissues. The extraction was based on an extraction followed by a concentration and clean-up step using weak cation exchange solid phase extraction. The separation was performed by ion-pair liquid chromatography on a C 18 column followed by mass spectrometric detection. The method was validated according to the EU requirements for a quantitative confirmatory method. Permethylated aminoglycosides (in-house synthesised internal standards) were used for accurate quantification. The accuracy of the analyses of AGs in kidney ranged from 94 to 111%, intra-day precision ranged between 2.5 and 7.4% (R.S.D. r) and inter-day precision ranged between 2.2 and 17.3% (R.S.D. RL, n = 21, MRL level). Accuracy (muscle tissue) varied from 83 to 128% with an intra-day precision between 2.2 and 17.3% (R.S.D. r, n = 7, MRL level). From the results it was concluded that the method was able to monitor MRL levels which ranged from 750 to 20,000 μg kg −1 for kidney and from 50 to 10,000 μg kg −1 for muscle tissue.

Determination of Selected Stimulants in Urine for Sports Drug Analysis by Solid Phase Extraction via Cation Exchange and Means of Liquid Chromatography-Tandem Mass Spectrometry

Stimulatory substances applied during competition possess a reasonable potential as performance enhancing agents and their misuse in elite sport has been frequently reported during the last few decades. An analytical method for the qualitative determination of selected stimulants containing a primary or secondary amine moiety in human urine for doping control purposes was developed. A rapid and highly specific procedure based on a sample preparation using weak cation exchange solid phase extraction (SPE-XCW) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a C6-Phenyl analytical column allowed the unambiguous identification of the target analytes down to low ng mL(-1) concentration levels. Validation provided recovery rates of better than 75%, precisions of less than 20% and a linear approximation in the required working range (10-750 ng mL(-1)) were obtained for 19 different target compounds. This method provides a rugged and highly specific alternative to the established method utilising gas or liquid chromatography after liquid-liquid extraction.

Quantitative Determination of Adrenaline and Noradrenaline in Urine Using Liquid Chromatography-Tandem Mass Spectrometry

The quantitative determination of adrenaline (A) and noradrenaline (N) in human urine provides a helpful tool for sports sciences as well as clinical purposes. A rapid and highly specific procedure based on an off-line sample preparation using weak cation exchange solid phase extraction (CX-SPE) followed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed that allowed the unambiguous identification of the target analytes at low (10 ng mL−1 A and N), medium and high (500 ng mL−1 A and N) physiological concentration levels. Recovery rates ranged from 104 to 111%, precisions of better than 15% and a linear approximation in the aimed working range were obtained for both compounds. Thus, this method provides a rugged and highly specific alternative to the commonly used electrochemical or fluorescence detection strategies. In- and out-of-competition catecholamine concentrations in urine samples of six professional German first division soccer players were determined demonstrating an obvious decrease of the N/A-ratio post-competition, compared to pre-competition and out-of-competition values providing a parameter for the estimation of physiological and psychological stress.

Pharmacokinetics and Mammary Elimination of Imidocarb in Sheep and Goats

The pharmacokinetics and mammary excretion of imidocarb dipropionate, a therapeutic/prophylactic agent against a variety of tick-borne hemoparasitic diseases in domestic animals, have been investigated in sheep and goats. A commercial formulation of imidocarb di-propionate was injected i.m. at a single dose of 3mg/kg of body weight in 7 mature lactating ewes and 8 lactating does in good health. Blood samples were collected for 48h after administration and milk samples were collected every 12h for 10 d. A weak cation-exchange solid-phase procedure was used to remove imidocarb from plasma. A hexane/isoamyl alcohol liquid-liquid procedure was adopted to extract the drug from the milk of sheep. The same method was used for goat milk after exposing the matrices to enzymatic digestion. The extracted samples were analyzed by HPLC. The i.m. disposition kinetics of imidocarb in the 2 species showed significant differences in the rate of elimination (0.0075±0.002 and 0.025±0.004 L/h in sheep and goats, respectively), being faster in ewes than in does. Nevertheless, a smaller area under the concentration-time curve (12.21±0.76 and 9.49±0.54μg/mL per h in sheep and goats, respectively), a larger volume of distribution (4.18±0.44 and 7.68±0.57 L/kg in sheep and goats, respectively), and a longer mean residence time (9.07±0.77 and 14.75±2.20h in sheep and goats, respectively) were found in goats, suggesting a more rapid and effective drug storage in tissues during the first 48h after the injection. The concentrations of imidocarb in milk of both species were higher than in plasma. However, a fast passage through the blood-milk barrier and a high storage of imidocarb were observed in the milk of ewes, whereas the drug concentrations were not as high nor was the extent of drug penetration from blood to milk as great in the milk of goats (AUCmilk 0–48/AUCplasma 0–48 = 2.5±0.45 and 1.26±0.27 in sheep and goat, respectively). Despite the differences in pharmacokinetic behavior, and considering the sensitivity of pathogens to imidocarb, the same dosage regimen can be used for clinical efficacy against Babesia spp. infection in both species. In contrast, the differences in depletion of imidocarb residue in milk and the large variability in mammary drug elimination found in goats suggests that great care should be taken in defining the withdrawal time in small ruminant dairy species.

The first identification of the rare cyanobacterial toxin, homoanatoxin-a, in Ireland

The first identification of the rare cyanobacterial neurotoxin, homoanatoxin-a, in Ireland is reported. A sensitive fluorimetric liquid chromatographic (LC) method was applied to the analysis of homoanatoxin-a in the low μg/l range. The analysis of the anatoxins in water samples required weak cation exchange solid phase extraction, fluorimetric derivatisation with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and isocratic reversed-phase LC. Confirmation of toxin identity was made using LC with electrospray mass spectrometry (MS) of the NBD-derivatised homoanatoxin-a as well as LC–MS/MS of the free toxin. Application of the fluorimetric LC protocol to examine cyanotoxins in 20 Irish lakes resulted in the detection of homoanatoxin-a at four locations, Lough Sillan (24 μg/l), Inniscarra Reservoir (34 μg/l), Lough Key (12 μg/l), Caragh Lake (1.4 μg/l). An improved procedure for the isolation of homoanatoxin-a from cyanobacteria was also developed and confirmation of homoanatoxin-a was achieved by chromatographic and mass spectral comparison with authentic toxin isolated from a laboratory clone culture of Planktothrix (formerly Oscillatoria) formosa.

Pharmacokinetics of imidocarb dipropionate in horses after intramuscular administration.

The objective of this study was to determine the pharmacokinetic behaviour of imidocarb in horses following a single i.m. injection at the dose commonly administered to treat Babesia caballi infections or to prevent babesiosis. Eight horses were injected i.m. with a single dose of 2.4 mg imidocarb dipropionate/kg bwt and blood, faecal, urine and milk samples were collected. For imidocarb determination, a high-performance liquid chromatographic method (HPLC) was used after weak cation-exchange solid phase, or liquid-liquid, extraction procedures. Twelve hours after treatment, no detectable plasma concentrations were recorded in any of the treated animals. The distribution and elimination patterns of the drug suggested that it is quickly sequestrated in some storage tissues and remains in the body for a long time. Its prolonged presence in the body may confer a reservoir effect to imidocarb in some tissues, therefore making it undetectable in the plasma of animals but sufficient to produce its described therapeutic and prophylactic activities.

Confirmation of gentamicin and neomycin in milk by weak cation-exchange extraction and electrospray ionization/ion trap tandem mass spectrometry.

A new procedure for the confirmation of two aminoglycoside antibiotics in milk was developed and validated. This work is among the early applications of ion trap mass spectrometry for regulatory methodology, and it incorporates a novel weak cation-exchange extraction. The procedure was validated for the confirmation of both gentamicin and neomycin at 30 ng ml(-1) and above. Milk is first treated with acid and centrifuged. The supernate, excluding the fat layer, is buffered with sodium citrate to neutral pH. The extract is applied to a weak cation-exchange solid-phase extraction column. Aminoglycosides are eluted with acidified methanol. Following separation by ion-pair liquid chromatography, analytes are ionized with an electrospray interface. Protonated molecular ions are selectively stored in an ion trap mass spectrometer, then collisionally dissociated to yield unique product ion spectra. Confirmation is based on matching spectral responses between samples and comparison standards consisting of a bona fide standard spiked into control extracts. Method performance was demonstrated with replicate samples of control milk, fortified milk, and milk containing incurred residues of each compound.

Plasma Sample Cleanup for Catecholamines Analysis Using Weak Cation Exchange Solid Phase Extraction

Abstract Norepinephrine and epinephrine were isolated from human plasma at picogram levels by solid phase extraction (SPE), on a proprietary weak cation exchange silica based packing. Plasma samples were spiked with catecholamines at two concentrations to simulate body response to varying levels of stress. Recoveries of norepinephrine and epinephrine at both concentrations exceeded 95%. An analysis of catecholamines by ion pair high-performance liquid chromatography with amperometric detection is described.