A stable-isotope HPLC-MS/MS method to simplify storage of human whole blood samples for glutathione assay.

BACKGROUND: Glutathione is the principal non-protein tripeptide thiol present in most mammalian cells and plays an important role in the redox status of biological systems. The accurate assessment of reduced glutathione (GSH) status as a reliable index of oxidative stress is of research and clinical significance. GSH undergoes rapid oxidation after sample collection and this presents a challenge. METHODS: Validation of an HPLC-MS/MS assay is reported. Storage stability using four variants of a methanolic precipitation with addition of stable isotope internal standard at collection is compared to L-serine borate/EDTA with perchloric acid precipitation (SBPE). RESULTS: Precipitation with methanol and addition of stable isotope on sample collection, combined with storage in solution at -70 °C showed superior storage stability to SBPE and other variants of the methanolic precipitation method up to 99 days. CONCLUSIONS: The combination of stable isotope with methanolic precipitation at collection, with assay by HPLC-MS/MS provides superior results after storage of whole blood samples for at least 99 days.

Comparative pharmacokinetics and pharmacodynamics of tablet, suspension and paste formulations of atenolol in cats.

This study compared the pharmacokinetic and pharmacodynamic profiles of an extemporaneously prepared (compounded) atenolol paste and suspension for oral administration, against the commercially available divided tablet in healthy cats. Eleven healthy cats (mean: age 4 ± 0.4 year, weight 5.0 ± 0.7 kg) were dosed twice-daily with 12.5 mg atenolol (tablet, paste or suspension) for 7 days in a randomized cross-over design with a 7-day wash-out period. On day 7, an electrocardiogram was performed before and immediately after stress provocation (jugular venipuncture) at prestudy screening, and at 2, 6 and 12 h after morning dosing. Systolic arterial blood pressure (BP) was assessed following the second electrocardiogram. Plasma was collected at prestudy screening, and at 1, 2, 6 and 12 h to measure atenolol plasma concentrations. Mean atenolol dose was 2.5 mg/kg (range: 2.1-3.3 mg/kg). Stress-induced rise in heart rate was attenuated (P < 0.05) at every time point compared to baseline for all formulations. Although the paste significantly attenuated stress-induced elevation in heart rate at all time points, the effect was not consistently equivalent to the tablet. The BP was not altered (P > 0.05) at any time point by any formulation. In conclusion, there were no significant differences (P > 0.05) in any of the pharmacokinetic parameters or pharmacodynamic profiles of the paste and suspension compared to the commercially available tablet.

A rapid and sensitive microscale HPLC method for the determination of indomethacin in plasma of premature neonates with patent ductus arteriousus.

Indomethacin (IND) is the drug of choice for the closure of a patent ductus arteriosus (PDA) in neonates. This paper describes a simple, sensitive, accurate and precise microscale HPLC method suitable for the analysis of IND in plasma of premature neonates. Samples were prepared by plasma protein precipitation with acetonitrile containing the methyl ester of IND as the internal standard (IS). Chromatography was performed on a Hypersil C(18) column. The mobile phase of methanol, water and orthophosphoric acid (70:29.5:0.5, v/v, respectively), was delivered at 1.5 mL/min and monitored at 270 nm. IND and the IS were eluted at 2.9 and 4.3 min, respectively. Calibrations were linear (r>0.999) from 25 to 2500 microg/L. The inter- and intra-day assay imprecision was less than 4.3 % at 400-2000 microg/L, and less than 22.1% at 35 microg/L. Inaccuracy ranged from -6.0% to +1.0% from 35 to 2000 microg/L. The absolute recovery of IND over this range was 93.0-113.3%. The IS was stable for at least 36 h when added to plasma at ambient temperature. This method is suitable for pharmacokinetic studies of IND and has potential for monitoring therapy in infants with PDA when a target therapeutic range for IND has been validated.

Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse.

Cylindrospermopsin (CYN) is a hepatotoxin isolated from the blue-green alga Cylindrospermopsis raciborskii. The role of both glutathione (GSH) and the cytochrome P450 enzyme system (P450) in the mechanism of toxicity of CYN has been previously investigated in in vitro systems. We have investigated the role of GSH and P450 in vivo in mice. Mice pre-treated with buthionine sulphoximine and diethyl maleate to deplete hepatic GSH prior to dosing with 0.2mg/kg CYN showed a seven-day survival rate of 5/13 while the control group rate was 9/14. Dosing mice with 0.2mg/kg CYN produced a small decrease in hepatic GSH with a characteristic rebound effect at 24h. The magnitude of this effect is however small and combined with the non-significant difference in survival rates after GSH depletion suggest depletion of GSH by CYN could not be a primary mechanism for CYN toxicity. Conversely, pre-treatment with piperonyl butoxide, a P450 inhibitor, protected mice against CYN toxicity giving a survival rate of 10/10 compared with 4/10 in the control group (p < 0.05 Chi squared) and was protective at doses up to 0.8 mg/kg, suggesting activation of CYN by P450 is of primary importance in the mechanism of action.

Extraction and purification of the zwitterions cylindrospermopsin and deoxycylindrospermopsin from Cylindrospermopsis raciborskii.

The hepatotoxin cylindrospermopsin (CYN) has been isolated from the cyanobacterium Cylindrospermopsis raciborskii (C. raci.). Efforts to study this toxin have been hampered by the time-consuming requirement to extract it from cultures of the organism. It is usually extracted from lyophilized cells collected from a laboratory culture. Our preliminary work suggested far more of the toxin is available in solution in the culture media than in the cells collected. We have therefore investigated the use of commercially available solid phase extraction sorbents to extract CYN from culture media in which C. raci. has been grown. A range of reverse phase and ion-exchange sorbents were tested across a range of pHs for their ability to retain CYN without success. Subsequently, graphitized carbon cartridges were found to retain CYN strongly. Elution with 5% formic acid in methanol allowed the CYN to be regained for final purification by HPLC. Deoxy-CYN, an analog of CYN can also be extracted using this procedure.

A sensitive and specific assay for glutathione with potential application to glutathione disulphide, using high-performance liquid chromatography-tandem mass spectrometry.

We have utilised the combination of sensitivity and specificity afforded by coupling high-performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS-MS) to produce an assay which is suitable for assaying glutathione (GSH) concentrations in liver tissue. The sensitivity suggests it may also be suitable for extrahepatic tissues. The method has been validated for GSH using mouse liver samples and also allows the assay of GSSG. The stability of GSH under conditions relevant to the assay has been determined. A 20-microl amount of a diluted methanol extract of tissue is injected with detection limits of 0.2 pmol for GSH and 2 pmol for GSSG. The HPLC uses an Altima C18 (150 x 4.6 mm, 5 microm) column at 35 degrees C. Chromatography utilises a linear gradient from 0 to 10% methanol in 0.1% formic acid over 5 min, with a final isocratic stage holding at 10% methanol for 5 min. Total flow rate is 0.8 ml/min. The transition from the M+H ion (308.1 m/z for GSH, and 613.3 m/z for GSSG) to the 162.0 m/z (GSH) and 355.3 m/z (GSSG) fragments are monitored.

Centipede envenomation.

Five episodes of envenomation by centipedes in 2 patients are reported. These arthropods are fast-moving, frightening in appearance to some, and may display aggressive behavior. However, stings from these centipedes, like most found worldwide, caused no serious morbidity or mortality. Common effects included intense local pain, erythema, induration, and necrosis, as well as mild constitutional symptoms. All resolved without sequelae. Treatment included pain control, wound care, and tetanus immunization.

Distribution of 14C cylindrospermopsin in vivo in the mouse.

Radiolabelled 14C cylindrospermopsin (CYN) has been prepared and used to investigate the distribution and excretion of CYN in vivo in male Quackenbush mice. At a dose of 0.2 mg/kg (i.e., approx. median lethal dose) the following mean (SD) urinary and faecal recoveries (cumulative) were obtained, respectively: (0-6 hours, n = 4) 48.2 (29.3)%, 11.9 (21.4)%; (0-12 hours, n = 12) 66.0 (27.1)%, 5.7 (5.6)%; (0-24 hours, n = 12) 68.4 (26.7)%, 8.5 (8.1)%. Mean (SD) recoveries from livers at 6 hours were 20.6 (6.4)% (n = 4), at 48 hours 13.1 (7.7)% (n = 8), and 5-7 days were 2.1 (2.1)% (n = 8). A substantial amount (up to 23%) can be retained in the liver for up to 48 hours with a lesser amount retained in the kidneys. The excretion patterns show substantial interindividual variability between predominantly faecal or urinary excretion, but these patterns are not related in any simple manner to the outcome in terms of toxicity. There is at least one methanol-extractable metabolite as well as a nonmethanol-extractable metabolite in the liver. The methanol-extractable metabolite was not found in the kidney and is more hydrophilic than CYN itself on reverse phase.

Airway management for the sports physician part 1: basic techniques.

Airway emergencies are, fortunately, rare in sports medicine, but when they occur, they must be addressed quickly and effectively. Various techniques can be applied by a trained team physician to optimize oxygenation and ventilation for an acutely ill or injured athlete. Initial management of airway emergencies on the field can be guided using a simple algorithm. Basic maneuvers include methods to clear airways, place ventilation devices, and assist with breathing. More advanced techniques include placing various endotracheal tube devices and performing surgical techniques; these will be discussed in a subsequent article.

Airway management for the sports physician part 2: advanced techniques.

Airway emergencies sometimes require techniques other than basic management methods. Advanced techniques are needed to manage laryngeal edema or fracture, upper-airway hemorrhage, or other injuries that make ventilation or intubation from above impossible. Placing various endotracheal devices and performing surgical techniques such as needle cricothyrotomy and tracheostomy can be done by physicians who have training and the necessary equipment. Surgical techniques can be performed with medical bag components, but commercial kits are available for those who are uncomfortable performing techniques using bag equipment.

Managing arthropod bites and stings.

Most arthropod bites and stings cause limited swelling, itching, pain, and redness and can be managed by ice application and tetanus prophylaxis as necessary. Stings by bees, wasps, and stinging ants can cause anaphylaxis that may require treatment with epinephrine and antihistamines and respiratory and cardiac maintenance measures. Widow spider bite management is controversial, but interventions for systemic reactions include calcium gluconate, methocarbamol, diazepam, narcotics, and antivenom. Victims of brown spider bites may need hospitalization if lesions enlarge rapidly or there are signs of systemic poisoning. Those stung by a bark scorpion may require oxygen, an intravenous line, pulse oximetry, and cardiac monitoring.

High-performance liquid chromatography-tandem mass spectrometry as a reference for analysis of tacrolimus to assess two immunoassays in patients with liver and renal transplants.

The accuracy and imprecision of three assays used for therapeutic monitoring of tacrolimus were tested using blood-containing weighed-in amounts of the drug, an enzyme-linked immunosorbent assay (ELISA), a microparticle enzyme immunoassay (MEIA I), and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) assay. Accuracy was acceptable for the HPLC-MS2 assay at all concentrations tested (< 10% deviation) and for the ELISA at 1.0 and 4.0 microg/l. Accuracy was not acceptable for the ELISA at 15.0 and 50.0 microg/l or for the MEIA I at all concentrations tested. Imprecision was acceptable for the HPLC-MS2 assay at all concentrations tested (coefficient of variation < 10%), for the ELISA at 15.0 and 50.0 microg/l, and for the MEIA I at 15.0 and 50.0 microg/l. Imprecision was not acceptable for the ELISA at 1.0 and 4.0 microg/l or for the MEIA I at 1.0 and 4.0 microg/l. This assessment with weighed-in amounts of tacrolimus verified the HPLC-MS2 assay as a reference method. The performance of the two immunoassays with HPLC-MS2 was then compared in the clinical setting using blood from patients with liver (n = 30) and renal (n = 37) transplants. In the liver transplant group (127 samples), the range of tacrolimus concentrations measured by HPLC-MS2, ELISA, and MEIA I was 1.9 to 31.8, 2.1 to 35.0, and less than 0.1 to 36.5 mg/l, respectively. In the renal transplant group (129 samples), the ranges were 1.7 to 26.1, 1.9 to 24.4, and 0.9 to 28.5 microg/l, respectively. Compared with the HPLC-MS2, the ELISA had minimal bias (0.1 to 0.2 microg/l) but unacceptable variability in values (SD > 13%). The MEIA I had unacceptable bias (1.7-1.8 microg/l) and variability (SD > 23%). These data indicated that neither the ELISA nor MEIA I is interchangeable with HPLC-MS2. Moreover, in view of the current trend to reduce the therapeutic dose of tacrolimus, quantitative results using the MEIA I would not be obtainable during therapeutic drug monitoring in some patients in whom effective therapeutic concentrations can be less than 5.0 microg/l.

Sensitive, specific quantitative analysis of tacrolimus (FK506) in blood by liquid chromatography-electrospray tandem mass spectrometry.

The capacity of liquid chromatography-tandem mass spectrometry (LC-MS2) to detect and define individual components in a complex mixture has been utilized to develop a quantitative assay of the potent immunosuppressant drug, tacrolimus. Trough blood concentrations were measured in 175 samples obtained over several weeks after liver transplantation from seven patients. The assay was linear over the range of 0.2 to 100 micrograms/L. Imprecision was <8%, and accuracy was 99-101%. The turnaround time for a batch of 20 samples was 2.5 h. No interference from any of the other drugs being administered to the patients was evident. An ELISA also performed on the same samples overestimated the concentrations substantially, as indicated by a plot of the difference between the results for the two methods vs their mean. The favorable characteristics of the LC-MS2 assay, especially its sensitivity and specificity, will facilitate detailed pharmacokinetic studies of tacrolimus, particularly under circumstances in which metabolism is perturbed by either hepatic dysfunction or drug interactions.

Evaluation and comparison of the TDxII, Stratus, and OPUS digoxin assays.

Three automated immunoassays for digoxin in serum were evaluated--Abbott TDxII, Baxter Stratus, and Behring OPUS. The accuracy and precision of the assays were assessed by weighed-in controls and an external quality control program. Coefficients of variation of all methods in serum were < or = 10% at weighed-in concentrations of digoxin of 1 and 2.5 micrograms/L. Accuracy relative to weighed-in concentrations of 1 and 2.5 micrograms/L ranged from 98 to 126% for all methods. Comparative results from patient samples showed little difference between the TDxII and Stratus and a greater difference observed between the TDxII and OPUS assays. The detection of digoxin-free samples containing digoxin-like immunoreactive substances (DLIS) in neonatal cord blood, pregnant patients, and liver and renal recipients by each assay was then assessed. The TDxII exhibited the highest incidence of DLIS. This is evident in neonatal cord blood in which 40.4% of samples tested positive. In comparison, the extent of DLIS detected by Stratus was less and OPUS exhibited no DLIS in any of the groups studied. A case study of a patient treated with anti-digoxin Fab fragments (Digibind) also was included for analysis by each method. Fourteen hours after Digibind administration, the TDxII registered a digoxin concentration of 49.5 micrograms/L compared with 3.73, 1.80, and 2.49 micrograms/L for Stratus, OPUS, and ultrafiltered TDxII methods, respectively. The results indicate that to determine the concentration of digoxin after the administration of Digiband, the OPUS or fluorescence polarization immunoassay (FPIA)-ultrafiltered samples by TDxII are the assays of choice.

Sensitive high-performance liquid chromatographic assay with ultraviolet detection of methadone enantiomers in plasma.

Methadone is being prescribed increasingly as an analgesic in palliative medicine. R-Methadone has been shown to be responsible for most of the pharmacological activity of this drug. Despite that in most countries it is administered as the racemate. Few assay methods for the enantiomers are available; e en fewer can determine accurately the low concentrations of enantiomers required to undertake pharmacokinetic studies in patients taking the drug in analgesic doses. We present here an HPLC method used to determine concentrations of the specific enantiomers of methadone as low as 5.0 ng/ml with adequate precision and accuracy. The mean R/S ratio of the plasma concentrations was 0.80 +/- 0.05 (n = 3 samples) in one patient taking 25-27.5 mg daily and 1.21 +/- 0.12 (n = 6 samples) in another taking 10-20 mg daily. In the second patient, concentrations of the enantiomers ranged between 5.8 and 25.9 ng/ml. Tricyclic antidepressants did not interfere with the assay but dextropropoxyphene did. Its presence could be detected by dual wavelength monitoring.