Formulation technologies for oral vaccines.

Many options now exist for constructing oral vaccines which, in experimental systems, have shown themselves to be able to generate highly effective immunity against infectious diseases. Their suitability for implementation in clinical practice, however, for prevention of outbreaks, particularly in low- and middle-income countries (LMIC), is not always guaranteed, because of factors such as cost, logistics and cultural and environmental conditions. This brief overview provides a summary of the various approaches which can be adopted, and evaluates them from a pharmaceutical point, taking into account potential regulatory issues, expense, manufacturing complexity, etc., all of which can determine whether a vaccine approach will be successful in the late stages of development. Attention is also drawn to problems arising from inadequate diet, which impacts upon success in stimulating effective immunity, and identifies the use of lipid-based carriers as a way to counteract the problem of nutritional deficiencies in vaccination campaigns.

Frequency, mutual exclusivity and clinical associations of myositis autoantibodies in a combined European cohort of idiopathic inflammatory myopathy patients.

OBJECTIVES: To determine prevalence and co-existence of myositis specific autoantibodies (MSAs) and myositis associated autoantibodies (MAAs) and associated clinical characteristics in a large cohort of idiopathic inflammatory myopathy (IIM) patients. METHODS: Adult patients with confirmed IIM recruited to the EuroMyositis registry (n = 1637) from four centres were investigated for the presence of MSAs/MAAs by radiolabelled-immunoprecipitation, with confirmation of anti-MDA5 and anti-NXP2 by ELISA. Clinical associations for each autoantibody were calculated for 1483 patients with a single or no known autoantibody by global linear regression modelling. RESULTS: MSAs/MAAs were found in 61.5% of patients, with 84.7% of autoantibody positive patients having a sole specificity, and only three cases (0.2%) having more than one MSA. The most frequently detected autoantibody was anti-Jo-1 (18.7%), with a further 21 specificities each found in 0.2-7.9% of patients. Autoantibodies to Mi-2, SAE, TIF1, NXP2, MDA5, PMScl and the non-Jo-1 tRNA-synthetases were strongly associated (p < 0.001) with cutaneous involvement. Anti-TIF1 and anti-Mi-2 positive patients had an increased risk of malignancy (OR 4.67 and 2.50 respectively), and anti-SRP patients had a greater likelihood of cardiac involvement (OR 4.15). Interstitial lung disease was strongly associated with the anti-tRNA synthetases, anti-MDA5, and anti-U1RNP/Sm. Overlap disease was strongly associated with anti-PMScl, anti-Ku, anti-U1RNP/Sm and anti-Ro60. Absence of MSA/MAA was negatively associated with extra-muscular manifestations. CONCLUSIONS: Myositis autoantibodies are present in the majority of patients with IIM and identify distinct clinical subsets. Furthermore, MSAs are nearly always mutually exclusive endorsing their credentials as valuable disease biomarkers.

The glucose lowering effect of an oral insulin (Capsulin) during an isoglycaemic clamp study in persons with type 2 diabetes.

AIM: Randomized, open, single-centre, two-way crossover study comparing the pharmacokinetic (PK) and pharmacodynamic (PD) properties of subcutaneous (sc) regular human insulin (Actrapid) and oral insulin in a capsule form (Capsulin). METHODS: Sixteen persons (12 males) with type 2 diabetes on oral hypoglycaemic agents (OHAs) participated. Mean (s.d.) age 60.2 (5.5) years, BMI 28.3 (3.4) kg/m(2), haemoglobin A(1c) (HbA(1c)) 7.4% (1.1). Two 6-h isoglycaemic glucose clamp studies were conducted 11 days apart. All subjects received in random order 12U sc Actrapid on one clamp study day and either 150U or 300U Capsulin (Cap) on the other day. Glucose infusion rates (GIRs), plasma insulin and C-peptide concentrations were determined throughout each 6-h isoglycaemic clamp. Between the clamp study days, all patients received 150U Capsulin twice daily, dropping all their standard OHAs apart from metformin. Self-monitored blood glucose (SMBG) levels were taken four times a day between the clamp study days. RESULTS: Administration of either Actrapid or Capsulin (150 and 300U) increased GIRs reaching a maximum values at approximately 280-330 min. Overall values for maximum GIR values were higher for Actrapid than either dose of Capsulin (p < 0.05). The significantly greater systemic insulin concentrations following Actrapid were reflected in the AUC(0-6 h) (910 +/- 270 vs. 472 +/- 245 pmol h/L; 950 +/- 446 vs. 433 +/- 218 pmol h/L; both p < 0.05 for Actrapid vs. 150U Capsulin and 300U Capsulin respectively). No difference was observed between 150U and 300U Capsulin. During the repeat-dosing period, good safety and tolerability were observed with Capsulin, and SMBG levels remained stable. At the poststudy visit, significant falls in HbA(1c), weight and triglycerides were observed. CONCLUSIONS: Administration of the oral insulin Capsulin preparation demonstrated a significant hypoglycaemic action over a period of 6 h associated with only a small increase in circulating plasma insulin concentrations.

Influence of the A and B subunits of cholera toxin (CT) and Escherichia coli toxin (LT) on TNF-alpha release from macrophages.

In this study an in vitro model was developed with the aim of investigating the modulatory effect of cholera toxin (CT) and its counterpart the heat labile toxin of Escherichia coli (LT) on TNF-alpha release induced by murine macrophages and primary human monocytes. Previous studies have demonstrated that the enzymatic activity of CT and LT molecules can inhibit TNF-alpha release by macrophages. The results obtained in this study showed that CT and LT are both, in a dose dependent manner, able either to induce or inhibit TNF-alpha release by murine macrophages and primary human monocytes. The results also showed that recombinant B subunits of CT and LT in the absence of their A subunit induce high levels of TNF-alpha release by macrophages and, in addition, increase the level of TNF-alpha release induced by LPS. The ability of both B subunits (CTB and LTB) in inducing TNF-alpha release by macrophages is not related to the level of LPS contamination, since direct measurements of LPS made in the samples employed in this study showed only traces of LPS (3.4 x 10(-8) EU/ml) which is in our system does not induce TNF-alpha release by macrophages. In contrast to the results obtained with the B subunits, incubation of cells with the A subunit of CT (CTA) inhibit TNF-alpha release induced by native CT, native LT, recombinant LTB and LPS. This inhibitory effect must be related to the activity of the A subunit since viability tests performed in terms of metabolic rate demonstrated that high concentrations of CTA are not toxic to the cells. The data presented herein demonstrate that the A subunits of CT and LT have an inhibitory effect on TNF-alpha release in macrophages, whereas their B subunits have a stimulatory effect on TNF-alpha. The results also suggest that the dose dependent bi-modal effect of native CT and native LT on TNF-alpha release by macrophages is a result of the combined effect of their individual A and B subunits.

Antibody-mediated protection against MERS-CoV in the murine model.

Murine antisera with neutralising activity for the coronavirus causative of Middle East respiratory syndrome (MERS) were induced by immunisation of Balb/c mice with the receptor binding domain (RBD) of the viral Spike protein. The murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the MERS coronavirus (MERS-CoV). To test the neutralising capacity of these antisera in vivo, susceptibility to MERS-CoV was induced in naive recipient Balb/c mice by the administration of an adenovirus vector expressing the human DPP4 receptor (Ad5-hDPP4) for MERS-CoV, prior to the passive transfer of the RBD-specific murine antisera to the transduced mice. Subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the MERS-CoV resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. The murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of RBD with a human IgG Fc tag (RBD-Fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. The data gained indicate that this dual-route vaccination with novel formulations of the RBD-Fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of MERS-CoV.

The high prevalence of unrecognized anaemia in patients with diabetes and chronic kidney disease: a population-based study.

BACKGROUND: Anaemia occurs early in the course of diabetes-related chronic kidney disease (CKD). There is little evidence about the prevalence of anaemia in people with diabetes. The aim of this study was to assess the prevalence of anaemia, by stage of CKD, in the general diabetic population. METHODS: Haemoglobin (Hb) was measured on all glycated haemoglobin (HbA1c) samples and the most recent (< 4 months) estimated glomerular filtration rate (eGFR) was obtained. Anaemia (at treatment level) was defined as Hb < 110 g/l or the use of erythropoetic stimulating agents (ESA). RESULTS: Twelve per cent (10-14%) of people had Hb < 110 g/l. The prevalence of anaemia increased progressively with worsening CKD. People with CKD stage 3 accounted for the largest number of people with anaemia; 18% (95% CI 13-24%) had Hb < 110 g/l. Those with eGFR < 60 ml/min/1.73 m2 and not on ESA or dialysis were four (2-7) times more likely than patients with better renal function to have Hb < 110 g/l. The relation between Hb and eGFR became approximately linear below an eGFR of 83 ml/min/1.73 m2, where, for every 1 ml/min/1.73 m2 fall in eGFR, there was a 0.4 (0.3-0.5) g/l fall in haemoglobin. CONCLUSIONS: This study demonstrates that anaemia, at levels where treatment is indicated, occurs commonly in people with diabetes and CKD stage 3 or worse. The screening for anaemia in current diabetes management should be extended.

Dual route vaccination for plague with emergency use applications.

Here, we report a dual-route vaccination approach for plague, able to induce a rapid response involving systemic and mucosal immunity, whilst also providing ease of use in those resource-poor settings most vulnerable to disease outbreaks. This novel vaccine (VypVaxDuo) comprises the recombinant F1 and V proteins in free association. VypVaxDuo has been designed for administration via a sub-cutaneous priming dose followed by a single oral booster dose and has been demonstrated to induce early onset immunity 14 days after the primary immunisation; full protective efficacy against live organism challenge was achieved in Balb/c mice exposed to 2 × 104 median lethal doses of Yersinia pestis Co92, by the sub-cutaneous route at 25 days after the oral booster immunisation. This dual-route vaccination effectively induced serum IgG and serum and faecal IgA, specific for F1 and V, which constitute two key virulence factors in Y. pestis, and is therefore suitable for further development to prevent bubonic plague and for evaluation in models of pneumonic plague. This is an essential requirement for control of disease outbreaks in areas of the world endemic for plague and is supported further by the observed exceptional stability of the primary vaccine formulation in vialled form under thermostressed conditions (40 °C for 29 weeks, and 40 °C with 75% relative humidity for 6 weeks), meaning no cold chain for storage or distribution is needed. In clinical use, the injected priming dose would be administered on simple rehydration of the dry powder by means of a dual barrel syringe, with the subsequent single booster dose being provided in an enteric-coated capsule suitable for oral self-administration.

Effects of combined angiotensin II and endothelin receptor blockade with developing heart failure: effects on left ventricular performance.

BACKGROUND: The goal of this study was to determine the comparative effects of angiotensin II type 1 (AT(1)) receptor inhibition alone, endothelin-1 (ET) receptor blockade alone, and combined receptor blockade on left ventricular (LV) function, contractility, and neurohormonal system activity in a model of congestive heart failure (CHF). METHODS AND RESULTS: Pigs were randomly assigned to each of 5 groups: (1) rapid atrial pacing (240 bpm) for 3 weeks (n=9), (2) concomitant AT(1) receptor blockade (valsartan, 3 mg/kg per day) and rapid pacing (n=8), (3) concomitant ET receptor blockade (bosentan, 50 mg/kg BID) and rapid pacing (n=8), (4) concomitant combined AT(1) and ET receptor inhibition and rapid pacing (n=8), and (5) sham-operated control (n=9). LV stroke volume was reduced from the control value after rapid pacing, was unchanged with either AT(1) or ET receptor blockade alone, but was improved with combination treatment. LV peak wall stress was reduced in both groups with ET receptor blockade compared with the rapid pacing group. Plasma norepinephrine levels were increased by >3-fold after rapid pacing, remained increased in the monotherapy groups, but were reduced after combination treatment. LV myocyte velocity of shortening was reduced after rapid pacing-induced CHF, remained reduced after AT(1) receptor blockade, increased after ET receptor blockade (compared with rapid pacing-induced CHF values), and returned to within control values after combined blockade. CONCLUSIONS: Combined AT(1) and the ET receptor blockade in this model of CHF improved LV pump function, and contributory factors included the effects of LV loading conditions, neurohormonal system activity, and myocardial contractile performance. Thus, combined receptor blockade may provide a useful combinatorial therapeutic approach in CHF.

Quantitative and ultrastructural studies on the uptake of drug loaded liposomes by mononuclear phagocytes infected with Leishmania donovani.

This study compared splenic and hepatic uptake of free and liposome-entrapped sodium antimony gluconate after i.v. administration to mice infected with Leishmania donovani. It was demonstrated that entrapment within liposomes greatly altered the kinetics of uptake of the drug. We were also able to show that liposomes composed of sphingomyelin, stearylamine and cholesterol were marginally better than any other preparation in delivering entrapped drug to liver and spleen. X-ray microanalytical studies on the uptake of liposomes by Kupffer cells infected with L. donovani have indicated that internalised liposomes probably fuse with parasitophorous vacuoles, transferring their contents into the immediate locality of the leishmanial parasites. It is proposed that this is the way in which liposome entrapped antileishmanial agents have an enhanced therapeutic effect over free drug therapy.

Specific unresponsiveness to skin allografts in mice. IV. Immunological reactivity of mice treated with liver extracts, Bordetella pertussis, and antilymphocyte serum.

The strain-specific unresponsiveness to H-2 incompatible skin allografts induced by treatment of adult mice with single inoculations of donor strain liver extract and Bordetella pertussis vaccine, as well as three doses of antilymphocyte serum, has been investigated by several in vivo and in vitro methods, with a view to elucidating it mechanism. Lymphoid cells from mice with long surviving skin grafts were found to be reactive in graft-versus-host assays (as measured by splenomegaly or popliteal lymph node enlargement), and mixed lymphocyte culture tests gave positive results. Attempts to cause lethal runting of F1 hybrid mice injected at birth with spleen cells from unresponsive mice gave variable results. However, the injection of F1 hybrid cells into the footpads of unresponsive animals failed to elicit a significant host-versus-graft response. Although lymphoid cells from unresponsive animals did not include detectable numbers of cytotoxic cells, such cells could be generated by previous in vitro mixed lymphocyte culture stimulation or, to some degree, by the injection of the animals with F1 hybrid cells. Attempts to prevent mixed lymphocyte culture stimulation or cytotoxicity with serum from unresponsive mice failed at the serum concentrations used. The data indicate that long-term unresponsiveness in this system is maintained by the production in the hosts of factors that interfere with the cell-mediated response.

Isolated left ventricular myocyte contractility in patients undergoing cardiac operations.

BACKGROUND: Because of methods required for obtaining isolated left ventricular myocytes, evaluation of the contractile function of isolated left ventricular myocytes in normal human patients has been limited. Accordingly, the goal of the present study was to develop a means to isolate human left ventricular myocytes from small myocardial biopsy specimens collected from patients undergoing elective coronary artery bypass operations and to characterize indices of myocyte contractile performance. METHODS: Myocardial biopsy specimens were obtained from the anterior left ventricular free wall of 22 patients undergoing coronary artery bypass operations. Myocytes were isolated from these myocardial samples by means of a stepwise enzymatic digestion method and micro-trituration techniques. Isolated left ventricular myocyte contractile function was assessed by computer-assisted high-speed videomicroscopy under basal conditions and in response to beta-adrenergic receptor stimulation with isoproterenol. RESULTS: A total of 804 viable left ventricular myocytes were successfully examined from all of the myocardial biopsy specimens with an average of 37+/-4 myocytes per patient. All myocytes contracted homogeneously at a field stimulation of 1 Hz with an average percent shortening of 3.7%+/-0.1% and shortening velocity of 51.3+/-1.3 microm/s. After beta-adrenergic receptor stimulation with isoproterenol, percent shortening and shortening velocity increased 149% and 118% above baseline, respectively (P < .05). CONCLUSION: The unique results of the present study demonstrated that a high yield of myocytes could be obtained from human left ventricular biopsy specimens taken during cardiac operations. These myocytes exhibited stable contractile performance and maintained the capacity to respond to an inotropic stimulus. The methods described herein provide a basis by which future studies could investigate intrinsic and extrinsic influences on left ventricular myocyte contractility in human beings.

Endothelin receptor pathway in human left ventricular myocytes: relation to contractility.

BACKGROUND: Increased synthesis and release of the potent bioactive peptide endothelin-1 (ET-1) occurs during and after cardiac surgery. However, the cellular and molecular basis for the effects of ET-1 on human left ventricular (LV) myocyte contractility remains unknown. METHODS: LV myocyte contractility was examined from myocardial biopsies taken from patients (n = 30) undergoing elective coronary artery bypass. LV myocytes (n = 997, > 30/patient) were isolated using microtrituration and contractility examined by videomicroscopy at baseline and after ET-1 exposure (200 pmol/L). In additional studies, myocytes were pretreated to inhibit either protein kinase C (PKC) (chelerythrine, 1 micromol/L), the sodium/hydrogen (Na/H) exchanger (EIPA, 1 micromol/L), both PKC and the Na/H exchanger, or the ET(A) receptor (BQ-123, 1 micromol/L), followed with ET-1 exposure. RESULTS: Basal myocyte shortening increased 37.8 +/- 6.3% with ET-1 (p < 0.05). Na/H exchanger, PKC, and dual inhibition all eliminated the effects of ET-1. Furthermore, ET(A) inhibition demonstrated that ET-1 effects on myocyte contractility were mediated through the ET(A) receptor subtype. CONCLUSIONS: ET-1 directly influences human LV myocyte contractility, which is mediated through the ET(A) receptor and requires intracellular activation of PKC and stimulation of the Na/H exchanger.

Albendazole chemotherapy for human cystic and alveolar echinococcosis in north-western China.

Human echinococcosis is highly endemic in north-western China; the main treatment is by surgery. In this paper, we report the results of chemotherapy with albendazole (ABZ), 15-20 mg/kg/d orally, for 30 d with intervals of 10 d between treatments for 3-6 courses. For multi-organ cystic echinococcosis (CE) and alveolar echinococcosis (AE), patients were given 12-18 courses of ABZ. Patients were divided into 4 groups: (i) ABZ surgery group, albendazole with surgery for 21 CE cases: (ii) non-ABZ surgery group, 80 CE cases treated by surgery alone; (iii) ABZ CE group, albendazole treatment alone in 58 CE cases, and (iv) ABZ AE group, 14 AE patients treated by albendazole and surgical intervention and 5 AE patients treated by albendazole alone. Twenty-seven of 34 (79.4%) cysts in group (i) patients showed increased necrotic changes and decreased viability of the cysts compared to group (ii). However, 10 of 84 (11.9%) cysts in group (ii) patients showed spontaneous evidence of necrosis at surgery. In group (iii), ABZ treatment alone was successful in 14 (24.1%), resulted in improvement in 29 (50%) and had no effect in 15 (25.9%) patients. Seven cases in group (iv) improved, with diminished size of lesions which were non-viable. The remaining 7 cases in group (iv) showed evidence of cyst viability at surgery; 2 could not be saved after a further 15 courses of albendazole. Of the five AE patients in group (iv) who received only ABZ, one improved, 2 stabilized, one deteriorated and one died. Albendazole chemotherapy, while not completely effective, has an important role in treatment of both cystic and alveolar echinococcosis.

Use of liposomes for protective immunisation in sheep against Echis carinatus snake venom.

Nigerian Echis carinatus venom incorporated into sphingomyelin-cholesterol liposomes stabilized with osmium tetroxide produced a rapid, sustained and protective immune response when injected i.v. into sheep. The response was similar to that reported earlier in mice. The implications of the findings are discussed in relation to the production of cheaper and more effective antivenoms. The possible use of immunostimulants for increasing the protective effect of antisera raised using this method are also considered.

Liposomal immunisation against snake venoms.

A method is described which produces a high, permanent antibody response following a single injection of venom. Animals (mice, rabbits, sheep) were given intravenous, subcutaneous or orally administered Nigerian Echis carinatus (carpet viper) venom which had been incorporated into sphingomyelin-cholesterol liposomes whose membranes had been stabilized by cross-linking adjacent molecules of sphingomyelin using osmium tetroxide. Before use the preparations were thoroughly dialysed to remove any unbound osmium tetroxide. Antibody levels were estimated using enzyme immunoassay. Venom treated in this way and administered i.v. or s.c. produced a powerful, sustained and protective antibody response lasting for the lifetime of a mouse. We also report the development of significant antibody responses after oral administration of liposome-entrapped but not free venom.

Use of liposomes for protective immunisation against Crotalus durissus (tropical rattlesnake) venom.

Crotalus durissus venom has been described as a weak antigen when injected in combination with Freund's complete adjuvant during the course of traditional methods of equine immunisation. Antibody production is slow and unpredictable, with a wide variation in individual responses. In this experimental study, C. durissus venom was incorporated into stabilised sphingomyelin-cholesterol liposomes both in the presence and absence of lipopolysaccharide immunostimulant and injected by both i.v. and s.c. routes into mice and rabbits. A rapid, sustained and protective immune response was obtained following a single injection of these preparations in mice. Antibody levels were estimated using enzyme-linked immunosorbent assay (ELISA), and the protective effect was evaluated by subsequent challenge with a subcutaneous minimum lethal dose of the venom. Results indicated that the immune response was significantly potentiated by the presence of immunostimulant in the venom liposomes. The use of C. durissus venom liposomes should be a useful tool for the immunisation of animals both in experimental and commercial procedures.

Influence of sphingomyelin and TNF-alpha release on lethality and local inflammatory reaction induced by Loxosceles gaucho spider venom in mice.

It is well known that Loxosceles venom induces local dermonecrosis in rabbits, guinea pigs and humans but not in mice, although, depending on the dose, Loxosceles venom can be lethal to mice. In this work we demonstrate that mice injected intradermally in the dorsal area of the back can survive a lethal dose of Loxosceles gaucho venom and also develop an inflammatory reaction (with infiltration of leukocytes shown by histological analysis) at the local injection site when the venom is co-administered with sphingomyelin. It was observed that more venom was retained for a longer period of time at the local injection site when venom was co-administered with sphingomyelin. The presence of exogenous sphingomyelin did not influence significantly the release of TNF-alpha induced by L. gaucho venom. These results suggest that the action of venom on sphingomyelin, producing ceramide phosphate, causes the development of an inflammatory reaction, which in turn traps the venom in the local area for a long period of time and does not allow it to disperse systemically in a dose sufficient to cause death. Our findings also indicate that the size and availability of the local sphingomyelin pool may be important in determining the outcome of Loxosceles envenoming in different mammalian species.

Effect of Loxosceles gaucho venom on cell morphology and behaviour in vitro in the presence and absence of sphingomyelin.

This study was performed to investigate whether the toxic effects of Loxosceles gaucho venom on cells might be exerted via stimulators of TNF-alpha release generated by sphingomyelinase D--a major component of the venom. It was demonstrated that L. gaucho venom alone is unable to induce TNF-alpha release by J774A.1 cells, while in the presence of exogenous sphingomyelin it induces a high level of TNF-alpha release which is significantly increased by incubation with non-inactivated serum. Ceramide phosphate also induces TNF-alpha release in J774A.1 cells, but (unlike sphingomyelin/sphingomyelinase) the level of release is not influenced by the presence or otherwise of non-inactivated serum. L. gaucho venom does not induce proliferation of J774A.1 cells and even at high concentrations it does not affect their viability. J774A.1 cells, which prior to venom treatment were elongated and clumped, round up after venom treatment, but, revert to their original morphology after incubation with fresh medium. TNF-alpha resistant MRC-5 cells and TNF-alpha sensitive MCF-7 cells are susceptible to the toxic effect of both L. gaucho venom and ceramide phosphate. The results obtained in this study demonstrate that exogenous sphingomyelin can modulate, in vitro, the release of TNF-alpha induced by L. gaucho venom in mouse macrophages. In addition, the results also indicate that ceramide phosphate and L. gaucho venom are toxic to several different cell types, via a variety of mechanisms, some, but not all, of which may involve TNF-alpha as an intermediary.

An evaluation of sample preparation techniques for the GC/MS analysis of urinary mercapturic acid conjugates.

Recovery rates of four different techniques for the preparation of human urine samples spiked with N-acetyl-S-benzyl-L-cysteine (BMA) were compared at three different spiking levels. At concentrations of 1,000 ppm and 1 ppm in the urine, recoveries of BMA were greatest (80-96%) using an ion pair phase transfer technique and a C18 solid-phase extraction (C18) technique while an acidic ethyl acetate extraction method yielded 67-69% recoveries and a quaternary amine solid-phase extraction technique showed poor recoveries (5-7%). At 10 ppb, quantitative recovery could only be determined for the C18 technique due to interferences from samples prepared using the other three techniques. The results indicate that the C18 sample preparation technique followed by GC/MS analysis using stable isotopically labeled internal standards provides a rapid and accurate method for quantitation of mercapturic acids at low-ppb levels in the urine.