Analytical progresses of the International Olympic Committee and World Anti-Doping Agency Olympic laboratories.

The Summer Olympic Games constitute the biggest concentration of human sports and activities in a particular place and time since 776 BCE, when the written history of the Olympic Games in Olympia began. Summer and Winter Olympic anti-doping laboratories, accredited by the International Olympic Committee in the past and the World Anti-Doping Agency in the present times, acquire worldwide interest to apply all new analytical advancements in the fight against doping in sports, hoping that this major human event will not become dirty by association with this negative phenomenon. This article summarizes the new analytical progresses, technologies and knowledge used by the Olympic laboratories, which for the vast majority of them are, eventually, incorporated into routine anti-doping analysis.

Transscleral Coulomb-controlled iontophoresis of methylprednisolone into the rabbit eye: influence of duration of treatment, current intensity and drug concentration on ocular tissue and fluid levels.

The major problems associated with the use of corticosteroids for the treatment of ocular diseases are their poor intraocular penetration to the posterior segment when administered locally and their secondary side effects when given systemically. To circumvent these problems more efficient methods and techniques of local delivery are being developed. The purposes of this study were: (1) to investigate the pharmacokinetics of intraocular penetration of hemisuccinate methyl prednisolone (HMP) after its delivery using the transscleral Coulomb controlled iontophoresis (CCI) system applied to the eye or after intravenous (i.v.) injection in the rabbit, (2) to test the safety of the CCI system for the treated eyes and (3) to compare the pharmacokinetic profiles of HMP intraocular distribution after CCI delivery to i.v. injection. For each parameter evaluated, six rabbit eyes were used. For the CCI system, two concentrations of HMP (62.5 and 150mg ml(-1)), various intensities of current and duration of treatment were analyzed. In rabbits serving as controls the HMP was infused in the CCI device but without applied electric current. For the i.v. delivery, HMP at 10mg kg(-1)as a 62.5mg ml(-1)solution was used. The rabbits were observed clinically for evidence of ocular toxicity. At various time points after the administration of drug, rabbits were killed and intraocular fluids and tissues were sampled for methylprednisolone (MP) concentrations by high pressure liquid chromatography (HPLC). Histology examinations were performed on six eyes of each group. Among groups that received CCI, the concentrations of MP increased in all ocular tissues and fluids in relation to the intensities of current used (0.4, 1.0 and 2.0mA/0.5cm(2)) and its duration (4 and 10min). Sustained and highest levels of MP were achieved in the choroid and the retina of rabbit eyes treated with the highest current and 10min duration of CCI. No clinical toxicity or histological lesions were observed following CCI. Negligible amounts of MP were found in ocular tissues in the CCI control group without application of current. Compared to i.v. administration, CCI achieved higher and more sustained tissue concentrations with negligible systemic absorption. These data demonstrate that high levels of MP can be safely achieved in intraocular tissues and fluids of the rabbit eye, using CCI. With this system, intraocular tissues levels of MP are higher than those achieved after i.v. injection. Furthermore, if needed, the drug levels achieved with CCI can be modulated as a function of current intensity and duration of treatment. CCI could therefore be used as an alternative method for the delivery of high levels of MP to the intraocular tissues of both the anterior and posterior segments.

Determination of trimipramine and its demethylated and hydroxylated metabolites in plasma by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method has been developed, for the determination of trimipramine (TRI), desmethyltrimipramine (DTRI), didesmethyltrimipramine (DDTRI), 2-hydroxytrimipramine (2-OH-TRI) and 2-hydroxydesmethyltrimipramine (2-OH-DTRI). The method includes two derivatization steps with trifluoroacetic acid anhydride and N-methyl-N-(tert.-butyldimethyl silyl)trifluoroacetamide and the use of an SE-54 capillary silica column. The limits of quantitation were found to be 2 ng/ml for DTRI and 4 ng/ml for all other substances. Besides, methods have been optimized for the hydrolysis of the glucuronic acid conjugated metabolites. This specific detection method is useful, as polymedication is a usual practice in clinical situations, and its sensitivity allows its use for single-dose pharmacokinetic studies.

Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column.

In addition to the importance of sample preparation and extract separation, MS detection is a key factor in the sensitive quantification of large undigested peptides. In this article, a linear ion trap MS (LIT-MS) and a triple quadrupole MS (TQ-MS) have been compared in the detection of large peptides at subnanomolar concentrations. Natural brain natriuretic peptide, C-peptide, substance P and D-Junk-inhibitor peptide, a full D-amino acid therapeutic peptide, were chosen. They were detected by ESI and simultaneous MS(1) and MS(2) acquisitions. With direct peptide infusion, MS(2) spectra revealed that fragmentation was peptide dependent, milder on the LIT-MS and required high collision energies on the TQ-MS to obtain high-intensity product ions. Peptide adsorption on surfaces was overcome and peptide dilutions ranging from 0.1 to 25 nM were injected onto an ultra high-pressure LC system with a 1 mm id analytical column and coupled with the MS instruments. No difference was observed between the two instruments when recording in LC-MS(1) acquisitions. However, in LC-MS(2) acquisitions, a better sensitivity in the detection of large peptides was observed with the LIT-MS. Indeed, with the three longer peptides, the typical fragmentation in the TQ-MS resulted in a dramatic loss of sensitivity (> or = 10x).

A LC-tandem MS assay for the simultaneous measurement of new antiretroviral agents: Raltegravir, maraviroc, darunavir, and etravirine.

Raltegravir (RAL), maraviroc (MVC), darunavir (DRV), and etravirine (ETV) are new antiretroviral agents with significant potential for drug interactions. This work describes a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of plasma drug levels. Single-step extraction of RAL, MVC, DRV, ETV and RTV from plasma (100 microl) is performed by protein precipitation using 600 microl of acetonitrile, after the addition of 100 microl darunavir-d(9) (DRV-d(9)) at 1000 ng/ml in MeOH/H(2)O 50/50 as internal standard (I.S.). The mixture is vortexed, sonicated for 10 min, vortex-mixed again and centrifuged. An aliquot of supernatant (150 microl) is diluted 1:1 with a mixture of 20 mM ammonium acetate/MeOH 40/60 and 10 microl is injected onto a 2.1 x 50 mm Waters Atlantis-dC18 3 microm analytical column. Chromatographic separations are performed using a gradient program with 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electrospray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring detection in the positive mode. The method has been validated over the clinically relevant concentrations ranging from 12.5 to 5000 ng/ml, 2.5 to 1000 ng/ml, 25 to 10,000 ng/ml, 10 to 4000 ng/ml, and 5 to 2000 ng/ml for RAL, MRV, DRV, ETV and RTV, respectively. The extraction recovery for all antiretroviral drugs is always above 91%. The method is precise, with mean inter-day CV% within 5.1-9.8%, and accurate (range of inter-day deviation from nominal values -3.3 to +5.1%). In addition our method enables the simultaneous assessment of raltegravir-glucuronide. This is the first analytical method allowing the simultaneous assay of antiretroviral agents targeted to four different steps of HIV replication. The proposed method is suitable for the Therapeutic Drug Monitoring Service of these new regimen combinations administered as salvage therapy to patients having experienced treatment failure, and for whom exposure, tolerance and adherence assessments are critical.

Circulating EM66 is a highly sensitive marker for the diagnosis and follow-up of pheochromocytoma.

We have previously demonstrated that measurement of tissue concentration of the novel secretogranin II-derived peptide EM66 may help to discriminate between benign and malignant pheochromocytomas. The aim of the present study was to characterize EM66 in plasma and urine of healthy volunteers and pheochromocytoma patients, in order to further evaluate the usefulness of this peptide as a circulating marker for the management of the tumors. HPLC analysis of plasma and urine samples demonstrated that the EM66-immunoreactive material coeluted with the recombinant peptide. In healthy volunteers, plasma and urinary EM66 levels were, respectively, 2.6 (1.9-3.7) ng/ml and 2.9 (1.9-4.6) ng/ml. In patients with pheochromocytoma, plasma EM66 levels were 10-fold higher than those of healthy volunteers (26.9 (7.3-44) ng/ml), and returned to normal values after removal of the tumor. In contrast, urinary EM66 levels were not significantly different from those of healthy volunteers (3.2 (2.2-3.9) ng/ml). Measurement of total or free plasma metanephrines and 24 hr urinary metanephrines in our series of patients revealed that these tests, taken separately, are less sensitive than the EM66 determination. Pheochromocytes in primary culture secreted high levels of EM66, suggesting that the chromaffin tumor was actually responsible for the increased plasma peptide concentrations in the patients. These data indicate that EM66 is secreted in the general circulation and that elevated plasma EM66 levels are correlated with the occurrence of pheochromocytoma. Thus, EM66 is a sensitive plasma marker that should be considered as a complementary tool in the management of pheochromocytoma.

Sensitive bioassay for determination of fluconazole concentrations in plasma using a Candida albicans mutant hypersusceptible to azoles.

The antifungal agent fluconazole (FLC) is widely used in clinical practice. Monitoring FLC levels is useful in complicated clinical settings and in experimental infection models. A bioassay using Candida pseudotropicalis, a simple and cost-effective method, is validated only for FLC levels ranging from 5 to 40 mg/liter. An extension of the analytical range is needed to cover most yeast MICs. A new bioassay in RPMI agar containing methylene blue was developed using C. albicans DSY1024, a mutant rendered hypersusceptible to FLC constructed by the deletion of the multidrug efflux transporter genes CDR1, CDR2, CaMDR1, and FLU1. Reproducible standard curves were obtained with FLC concentrations in plasma ranging from 1 to 100 mg/liter (quadratic regression coefficient > 0.997). The absolute sensitivity was 0.026 microg of FLC. The method was internally validated according to current guidelines for analytical method validation. Both accuracy and precision lied in the required +/-15% range. FLC levels measured by bioassay and by high-performance liquid chromatography (HPLC) performed with 62 plasma samples from humans and rats showed a strong correlation (coefficients, 0.979 and 0.995, respectively; percent deviations of bioassay from HPLC values, 0.44% +/- 15.31% and 2.66% +/- 7.54%, respectively). In summary, this newly developed bioassay is sensitive, simple, rapid, and inexpensive. It allows nonspecialized laboratories to determine FLC levels in plasma to within the clinically relevant concentration range and represents a useful tool for experimental treatment models.

Improved Asphalt Surfaces and Asphalt Resurfacing Performance through Crack Maintenance, HR-213, 1987

In 1980, a Vanguard High Pressure Water Blaster capable of providing 10 gallons of water per minute at 2000 psi was purchased to evaluate water blasting as a crack cleaning method prior to crack filling on asphalt concrete pavements. Afer some iniital trials demonstrated its effectiveness of removing dirt, debris and vegetation, it was included in joint and crack maintenance research on Iowa 7 in Webster County. The objective of the research was to evaluate six crack preparation methods and seven "sealant" materials. The cleaning and sealing was performed in the spring of 1983. Visual evaluations of the performance were made in the fall of 1983 and spring of 1985. Compressed air and/or high pressure water did not adequately prepare cracks less than 3/8 inch wide. Routing or sawing was necessary to provide a sealant reservoir. The water blaster was more effective than compressed air in removing dirt, debris and vegetation but this did not yield significant improvement in sealant adhesion or longevity. Periodic crack filling is necessary on ACC surfaces throughout the remaining life of the pavement.

Image Analysis for Evaluating Air Void Parameters of Concrete, HR-396, 1998

This research project investigated the use of image analysis to measure the air void parameters of concrete specimens produced under standard laboratory conditions. The results obtained from the image analysis technique were compared to results obtained from plastic air content tests, Danish air meter tests (also referred to as Air Void Analyzer tests), high-pressure air content tests on hardened concrete, and linear traverse tests (as per ASTM C-457). Hardened concrete specimens were sent to three different laboratories for the linear traverse tests. The samples that were circulated to the three labs consisted of specimens that needed different levels of surface preparation. The first set consisted of approximately 18 specimens that had been sectioned from a 4 in. by 4 in. by 18 in. (10 cm by 10 cm by 46 cm) beam using a saw equipped with a diamond blade. These specimens were subjected to the normal sample preparation techniques that were commonly employed by the three different labs (each lab practiced slightly different specimen preparation techniques). The second set of samples consisted of eight specimens that had been ground and polished at a single laboratory. The companion labs were only supposed to retouch the sample surfaces if they exhibited major flaws. In general, the study indicated that the image analysis test results for entrained air content exhibited good to strong correlation to the average values determined via the linear traverse technique. Specimens ground and polished in a single laboratory and then circulated to the other participating laboratories for the air content determinations exhibited the strongest correlation between the image analysis and linear traverse techniques (coefficient of determination, r-squared = 0.96, for n=8). Specimens ground and polished at each of the individual laboratories exhibited considerably more scatter (coefficient of determination, r-squared = 0.78, for n=16). The image analysis technique tended to produce low estimates of the specific surface of the voids when compared to the results from the linear traverse method. This caused the image analysis spacing factor calculations to produce larger values than those obtained from the linear traverse tests. The image analysis spacing factors were still successful at distinguishing between the frost-prone test specimens and the other (more durable) test specimens that were studied in this research project.

The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.

There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing. For transfer, handling and subsequent milling, the tubes are placed in a novel connective device (ferrule) that protects the sample from devitrification and contamination and passes through all operation steps. A piezo driven sample positioning stage (cryo-nano-bench, CNB) with three degrees of freedom was additionally developed to enable accurate milling of frozen-hydrated lamellas. With the CNB, high-pressure frozen samples can be milled to produce either thin lamellas (<100 nm), for direct imaging by high-resolution cryo-TEM or thicker lamellas (300-500 nm) for cryo-electron tomography. The sample remains vitreous throughout the process by using the presented tools and methods. The results are an important step towards investigating larger cells and even tissue in there natural state which in the end will enable us to gain better insights into cellular processes.

Determination of the enantiomers of thioridazine, thioridazine 2-sulfone, and of the isomeric pairs of thioridazine 2-sulfoxide and thioridazine 5-sulfoxide in human plasma.

Thioridazine is a commonly prescribed phenothiazine drug administered as a racemate and it is believed that its antipsychotic effect is mainly associated with (R)-thioridazine. A method based on high-performance liquid chromatography has been developed for the determination of the enantiomers of thioridazine and thioridazine 2-sulfone (THD 2-SO2 or sulforidazine) and of the enantiomers of the diastereoisomeric pairs of thioridazine 2-sulfoxide (THD 2-SO or mesoridazine) and thioridazine 5-sulfoxide (THD 5-SO) in the plasma of thioridazine-treated patients. The method involves sequential achiral and chiral HPLC. The limits of quantitation for total (R) + (S) concentrations were found to be 15 ng/ml for thioridazine and 5 ng/ml for its metabolites. The limits for the determination of the (R)/(S) ratios were found to be 60 ng/ml for racemic THD and 10 ng/ml for racemic THD 2-SO, THD 2-SO2, THD 5-SO (FE) and THD 5-SO (SE). The method has been used to determine the concentrations of the enantiomers of thioridazine and of its metabolites in the plasma of a patient treated with 100 mg of racemic thioridazine hydrochloride per os per day for 14 days. The results show a high enantioselectivity in the metabolism of this drug: the (R)/(S) ratios for THD, THD 2-SO (FE), THD 2-SO (SE), THD 2-SO2, THD 5-SO (FE) and THD 5-SO (SE) were found to be 3.90, 1.22, 6.10, 4.10, 0.09 and 28.0, respectively.

Proteasomal cleavage does not determine immunogenicity of gp100-derived peptides gp100 209-217 and gp100 209-217T210M.

Immune responses against tumor-associated antigens rely on efficient epitope presentation. The melanoma-associated antigen (Ag) gp100 contains HLA-A*0201 ligands that are characterized by low to medium binding affinity, among which gp100(209-217) is the most prominent (Kawakami et al., J Immunol 154:3961-3968, 1995). While this epitope is a natural T-cell target, it primes with low-efficiency T-cell responses during immunization. A modified gp100 epitope, gp100(209-217T210M), that contains a Thr to Met substitution at position 2 of the antigenic nonamer is characterized by high binding affinity for HLA-A*0201 and elicits strong and clinically effective T-cell responses. This higher affinity is believed to represent the sole reason for enhanced immunogenicity. Contrasting with this observation is the unpredictable relationship between affinity and immunogenicity observed in other antigen systems. In addition, we noted a striking difference between the capability of endogenously processed gp100(209-217) and gp100(209-217T210M) to induce T-cell responses in an in vitro model. Therefore, we questioned whether factors other than HLA-affinity might play a role in determining the immunogenicity of these epitopes. In the present study, we evaluated the in vitro proteasomal cleavages of 23meric precursor peptides encompassing the native sequence (gp100(201-223)) or the modified sequence (gp100(201-223T210M)). Here we show that the standard proteasome liberates the C-termini of both antigenic peptides but not the N-termini. Quantitative analysis of the digestion products revealed that more of the fragments displaying the final C-termini were produced from the wild-type precursor. However, a stronger TCR engagement was observed when fractions of digested gp100(201-223T210M) were used to activate an HLA-A*0201-expressing target T-cell clone. This difference was also found using separately produced, synthetic nonamers. In conclusion, the high binding affinity of gp100(209-217T210M) seems to compensate for possible differences in proteasomal cleavage at the biological level. Since the final antigenic nonamer is not directly produced by the proteasome, additional further factors may influence the antigenic peptide availability, such as post-proteasomal processing and intracellular peptide transport.

On-line desorption of dried blood spot: A novel approach for the direct LC/MS analysis of micro-whole blood samples.

The aim of this work is to present a new concept, called on-line desorption of dried blood spots (on-line DBS), allowing the direct analysis of a dried blood spot coupled to liquid chromatography mass spectrometry device (LC/MS). The system is based on an inox cell which can receive a blood sample (10 microL) previously spotted on a filter paper. The cell is then integrated into LC/MS system where the analytes are desorbed out of the paper towards a column switching system ensuring the purification and separation of the compounds before their detection on a single quadrupole MS coupled to atmospheric pressure chemical ionisation (APCI) source. The described procedure implies that no pretreatment is necessary in spite the analysis is based on whole blood sample. To ensure the applicability of the concept, saquinavir, imipramine, and verapamil were chosen. Despite the use of a small sampling volume and a single quadrupole detector, on-line DBS allowed the analyses of these three compounds over their therapeutic concentrations from 50 to 500 ng/mL for imipramine and verapamil and from 100 to 1000 ng/mL for saquinavir. Moreover, the method showed good repeatability with relative standard deviation (RSD) lower than 15% based on two levels of concentration (low and high). Function responses were found to be linear over the therapeutic concentration for each compound and were used to determine the concentrations of real patient samples for saquinavir. Comparison of the founded values with those of a validated method used routinely in a reference laboratory showed a good correlation between the two methods. Moreover, good selectivity was observed ensuring that no endogenous or chemical components interfered with the quantitation of the analytes. This work demonstrates the feasibility and applicability of the on-line DBS procedure for bioanalysis.

Nanoparticles in SiH4-Ar plasma: Modelling and comparison with experimental data

Experimental and theoretical investigations for growth of silicon nanoparticles (4 to 14 nm) in radio frequency discharge were carried out. Growth processes were performed with gas mixtures of SiH4 and Ar in a plasma chemical reactor at low pressure. A distinctive feature of presented kinetic model of generation and growth of nanoparticles (compared to our earlier model) is its ability to investigate small"critical" dimensions of clusters, determining the rate of particle production and taking into account the influence of SiH2 and Si2Hm dimer radicals. The experiments in the present study were extended to high pressure (≥20 Pa) and discharge power (≥40 W). Model calculations were compared to experimental measurements, investigating the dimension of silicon nanoparticles as a function of time, discharge power, gas mixture, total pressure, and gas flow.

Determination of the enantiomers of mianserin, desmethylmianserin, and 8-hydroxymianserin in the plasma and urine of mianserin-treated patients.

An HPLC method is presented which allows the measurement in the same run of the enantiomers of mianserin, desmethylmianserin, and 8-hydroxymianserin in plasma and urine of mianserin-treated patients. Limits of quantitation for the (S)- and (R)-enantiomers of mianserin and desmethylmianserin were 4 and 2.5 ng/ml, respectively, in plasma, and for the (S)- and (R)-enantiomers of mianserin, desmethylmianserin, and 8-hydroxymianserin 5, 2.5, and 5 ng/ml, respectively, in urine. The measured ratios of (S)-mianserin/(R)-mianserin and (S)-desmethylmianserin/(R)-desmethylmianserin in the plasmas of 10 mianserin-treated patients, all extensive metabolizers of debrisoquine as determined by CYP2D6 genotyping, varied, respectively, from 1.0 to 4.06 and from 0.19 to 0.64. As the enantiomers of mianserin differ in their pharmacological profile, these results could partially explain why, until now, no consistent relationship has been established between the therapeutic response and total [(S) + (R)] plasma levels of this antidepressant.