Sweat testing for cocaine, codeine and metabolites by gas chromatography-mass spectrometry.

Sweat testing for drugs of abuse provides a convenient and considerably less invasive method for monitoring drug exposure than blood or urine. Numerous devices have been developed for collection of sweat specimens. The most common device in current use is the PharmChek Sweat Patch, which usually is worn by an individual for five to ten days. This device has been utilized in several field trials comparing sweat test results to conventional urinalysis and the results have been favorable. Two new Fast Patch devices have been developed and tested that allow rapid collection of sweat specimens. The Hand-held Fast Patch was applied to the palm of the hand and the Torso Fast Patch was applied to the abdomen or the sides of the trunk (flanks) of volunteer subjects participating in a research study. Both patches employed heat-induced sweat stimulation and a larger cellulose pad for increased drug collection. Sweat specimens were collected for 30 min at various times following administration of cocaine or codeine in controlled dosing studies. After patch removal, the cellulose pad was extracted with sodium acetate buffer, followed by solid-phase extraction. Extracts were derivatized and analyzed by gas chromatography mass spectrometry (GC-MS) simultaneously for cocaine, codeine and metabolites. Cocaine and codeine were the primary analytes detected in sweat. Peak cocaine and codeine concentrations ranged from 33 to 3579 ng/patch and 11 to 1123 ng/patch, respectively, across all doses for the Hand-held Patch compared to 22-1463 ng/patch and 12-360 ng/patch, respectively, for the Torso Fast Patch. Peak concentrations generally occurred 4.5-24 h after dosing. Both drugs could be detected for at least 48 h after dosing. Considerably smaller concentrations of metabolites of cocaine and codeine were also present in some patches. Generally, concentrations of cocaine and codeine were higher in sweat specimens collected with the Hand-held Fast Patch than for the Torso Fast Patch. Drug concentrations were also considerably higher than those reported for the PharmChek Sweat Patch. The predominance of cocaine and codeine in sweat over metabolites is consistent with earlier studies of cocaine and codeine secretion in sweat. Multiple mechanisms appear to be operative in determining the amount of drug and metabolite secreted in sweat including passive diffusion from blood into sweat glands and outward transdermal migration of the drug. Additional important factors are the physico-chemical properties of the drug analyte, specific characteristics of the sweat collection device, site of sweat collection and, in this study, the application of heat to increase the amount of drug secreted.

A collection method and high-sensitivity enzyme immunoassay for sweat pyridinoline and deoxypyridinoline cross-links.

BACKGROUND: Collagen cross-link molecules such as pyridinoline (PYD), deoxypyridinoline (DPD), and N-terminal cross-linked peptides (NTX) have been measured in urine as indices of bone resorption. However, very little is known regarding the excretion of pyridinolines into other biological fluids. We report a collection device, normalizing analyte, and high-sensitivity immunoassay for quantitative analysis of free pyridinoline cross-links in sweat. METHODS: Flame atomic emission and ion-selective electrode techniques were used to measure potassium as a sweat volume marker. The Pyrilinks immunoassay for urine free pyridinolines was optimized to increase sensitivity for measurements in sweat. The precision, accuracy, and detection limit of this assay were characterized. To assess values and variability of sweat pyridinolines in human subjects, a nonocclusive skin patch was used to collect sweat samples from a reference group and from a mixed group experiencing accelerated bone resorption, postmenopausal women and men receiving gonadotropin-releasing hormone for prostate cancer. RESULTS: The immunoassay intra- and interassay variations were

Caffeine demethylation monitoring using a transdermal sweat patch.

Caffeine and two metabolites (paraxanthine and theobromine) were quantitated by high-performance liquid chromatography (HPLC) using extracts from transdermal sweat patches that continuously collected and stored analytes lost through the skin. Following caffeine consumption, remarkably clean chromatograms were obtained with minimal sample preparation. Caffeine and paraxanthine accumulated in the patch at comparable rates, and theobromine accumulated more slowly. A major urinary metabolite, 1-methylxanthine, was notably absent in sweat-patch and plasma extracts, a finding which favors a renal source for this metabolite. A simple, noninvasive approximation of N-demethylation can be made by calculating the paraxanthine/ caffeine and theobromine/caffeine ratios in the patch extract. These ratios were significantly reduced in high-dose (600 mg) versus low-dose (200 mg) subjects, possibly reflecting a decreased clearance of caffeine. Because the sweat patches can be worn for several days, the technique gives a multiday historical record which reflects the fluctuating systemic concentration of caffeine and its hepatic metabolites and thus might be useful to noninvasively monitor compliance by caffeine-restricted patients or to assess drug-metabolizing status.

Qualitative detection of opiates in sweat by EIA and GC-MS.

Sweat was collected with the PharmChek sweat patch, and drugs were eluted from the collection pad of the patch. A solid-phase enzyme immunoassay (EIA) using microtiter plates was modified for the analysis of opiates in sweat. After opiate administration, sweat contains primarily parent opiate (heroin, codeine) and lipophilic metabolites (6-monoacetylmorphine [6-MAM]). The immunoassay was determined to have a cross-reactivity with codeine of 588%, with hydrocodone of 143%, with diacetylmorphine of 28%, and with 6-MAM of 30% relative to 100% for the morphine calibrators. The optimum cutoff concentration for this modified assay was determined by receiver operator characteristic analysis using 215 patches from 95 subjects to be 10 ng/mL morphine equivalents. At this cutoff concentration the assay had a diagnostic sensitivity of 86.9% and a diagnostic specificity of 92.8% versus gas chromatography-mass spectrometry (GC-MS), which was the reference method. The positive predictive value at a prevalence of 50% was 86%. The intra-assay precision at 10 ng/mL was 7.8%, and the interassay coefficient of variation (CV) was 39%. Analysis of spiked patches around the cutoff gave a percent positive threshold of approximately 50% between 10 and 15 ng/mL and a 95% confidence level for a positive result by the EIA between 20 and 25 ng/mL. Eighteen possible adulterants that could be injected into or under the patch were studied. Two (tile cleaner and detergent) can cause false-positive responses in the immunoassay. Two adulterants reduced response to spiked drug (Visine eye drops and Ben Gay ointment), which could cause a false-negative response. All results were confirmed by GC-MS. The clinical sensitivity and specificity for detecting drug use by analyzing sweat collected from human subjects following known doses of codeine (0, 30, and 60 mg orally) or heroin (20 mg intravenously) were 76 and 100%, respectively.

Detection of methamphetamine in sweat by EIA and GC-MS.

Sweat was collected with the PharmChekTM sweat patch and drugs were eluted from the collection pad of the patch. A solid phase, enzyme immunoassay using microtiter plates was modified for analysis of methamphetamine in sweat. After methamphetamine administration, sweat contains primarily parent methamphetamine. The immunoassay was determined to have crossreactivity relative to 100% for the methamphetamine (MA) calibrators; to 144% for methylenedioxymethamphetamine (MDMA); to 30% for d-amphetamine; to 21% for methylenedioxyamphetamine (MDA); and to 8% for I-methamphetamine. The optimum cutoff concentration for this modified assay was determined by receiver operating characteristic analysis to be 10 ng/mL amphetamine equivalents. At this cutoff concentration the assay had a diagnostic sensitivity of 84.5% and a diagnostic specificity of 93.2% versus gas chromatography-mass spectrometry (GC-MS). The positive predictive value at a prevalence of 50% was 86%. The intra-assay precision at 10 ng/mL was 9.9% (coefficient of variation, CV) and the interassay CV was 13%. Analysis of spiked patches at plus or minus 25 and 50% around the cutoff gave a percent positive threshold of approximately 50% at a cutoff of 10 ng/mL and a 95% confidence level for a positive result by the EIA between 15 and 20 ng/mL. Of 18 potential adulterants that might be injected into or under the patch, two (tile cleaner and cough syrup) caused a false-positive response by immunoassay. All results were confirmed by GC-MS. The clinical sensitivity and specificity of the overall analysis system (sweat collection and analysis) were 85 and 100%, respectively, using known methamphetamine dosing of volunteers (10, 20, and 25 mg) as the reference standard.

Enzyme immunoassay validation for qualitative detection of cocaine in sweat.

A solid-phase enzyme immunoassay (EIA) involving microtiter plates was modified for analysis of cocaine in sweat. Sweat was collected with the PharmChek sweat patch and drugs were eluted from the collection pad of the patch. The sweat contained primarily parent cocaine. The assay was determined to have cross-reactivity for cocaine of 102% relative to 100% for the benzoylecgonine (BE) calibrators and for cocaethylene of 148%. The optimum cutoff concentration for this modified assay, determined by receiver-operating characteristic curve analysis, was 10 micrograms/L cocaine or BE equivalents. At this concentration the assay had 94.5% sensitivity and 99.1% specificity vs gas chromatography-mass spectrometry (GC-MS) as an acceptable indicator of the true clinical state. The positive predictive value at a prevalence of 50% was 99%. Threshold analysis for positives suggested that the 95% confidence interval for a positive result by the EIA was between 12.5 and 15 micrograms/L and that quality-control samples at 5 and 15 micrograms/L could be run with each batch to certify the precision around the cutoff. All positive samples must be confirmed by GC-MS. The sensitivity and specificity of the overall analysis system (immunoassay screen and GC-MS confirmation) was 86% and 97%, with known cocaine dosing of volunteers as the acceptable indicator of the true clinical state.

Storage and transfusion of platelets collected by an automated two-stage apheresis procedure.

Platelets collected by using a two-stage automated blood cell separator were evaluated after 5 days of storage. The procedure caused no unanticipated physiologic changes in donors and produced greater than 3 x 10(11) intact platelets in 200 mL of plasma, plus an additional 400 mL of plasma with intact coagulation factors. Posttransfusion recovery of autologous radiolabeled platelets was comparable to that seen in platelets prepared by manual centrifugation techniques. Corrected count increments in 14 patients showed results similar to those with control transfusions. This device, which involves a collection time of less than 90 minutes, provides an option for platelet-pheresis in a variety of settings including blood mobiles.

Platelet collection with the Autopheresis-C apheresis system.

This report describes a new system for collection of platelet concentrate (PC) and cell-free plasma (PPP) from apheresis donors. The system uses two separation devices and requires only a single venipuncture. The Plateletcell device separates primary platelet concentrate (PPC) from anticoagulated whole blood and the Plasmacell-C device separates the PPC into PC and PPP. Results of functional studies performed indicate that the separation process does not alter viability of either the PPC, the PC, or the PPP. Platelet function after 5 days of storage is maintained. An average yield of 3.4 +/- 0.7 x 10(11) platelets in 201 g of PC and 422 g of PPP were harvested in 71 +/- 13 min of donor time from donors with preprocedure hematocrits averaging 42.5 +/- 2.0% and preprocedure platelet counts averaging 265 +/- 61 x 10(3)/microliters.

Formation of a cryogel during processing of cell-free plasma.

Automated plasmapheresis devices are being integrated into many modern plasma procurement programs. Owing to the use of a different concentration and type of anticoagulant, the recovered plasma differs in pH and citrate levels from that obtained by manual plasmapheresis or whole blood donation. Recently, fractionators noted the recovery of a sticky, gelatinous fraction (cryogel) during thawing of cell-free (CF) plasma, along with reduced recovery of factor VIII:C (FVIII:C) in the cryoprecipitate fraction. Following their manufacturing procedures, it was established that the cryogel fraction of CF plasma is enriched in FVIII:C, fibrinogen, and fibronectin, as compared to cryoprecipitate from CPDA-1 plasma. Cryogel formation was not significantly affected by pH or citrate adjustment of the recovered plasma, by the use of polycarbonate or nylon filter membranes, or by the filter wetting agent polyvinylpyrrolidone (PVP). Furthermore, passage of CPDA-1 plasma through the polycarbonate filter did not alter cryoprecipitate quality. However, cryogel formation from CF plasma was reduced significantly by 1) slow thawing at 4 degrees C rather than quick thawing at 20 and 0 or 20 and 4 degrees C, 2) the use of 1:16.6 sodium citrate rather than 1:12.5 ACD-A, or 3) the addition of intact platelets, platelet lysate, membranes, or cytosol to CF plasma before freezing. The data suggest an important and, indeed, essential role of platelet constituents in the formation of both cryoprecipitate and cryogel during the low-temperature purification of plasma proteins.

The surge technique: a method to increase purity of platelet concentrates obtained by centrifugal apheresis.

A surge technique has been developed to increase the purity of high-yield platelet concentrations prepared on a blood processor with the Latham bowl. The surge technique combines elutriation with centrifugal separation, utilizing plasma recirculated from the plasma/air bag back into the centrifuge bowl to elute platelets from the red cell mass. Platelet concentrates prepared by surge collection with six separation cycles (n = 22), contained an average of 3.9 +/- 1.4 X 10(11) platelets, with 0.15 +/- 0.11 X 10(9) leukocytes, and red cells below the level of detection. The surge technique reduces collection time by 4 minutes per cycle and eliminates the need for a secondary centrifugation; thus, 96 minutes donor processing time would permit eight separation cycles. Platelets collected by the surge technique exhibited unaltered morphology and capacity to take up radioactively labeled serotonin in vitro compared to pre-apheresis controls. Results from in vitro functional studies also indicate that the capacity of platelets collected with the surge technique to respond to various concentrations of adenosine diphosphate, collagen, and thrombin by aggregation and secretion of both serotonin (dense bodies) and beta-thromboglobulin (alpha-granules) was not significantly different (p less than or equal to 0.05) from that of pre-apheresis controls.