Severe hypercholesterolemia mediated by lipoprotein X in patients with chronic graft-versus-host disease of the liver.

We describe a series of cases of extreme hypercholesterolemia mediated by lipoprotein X in patients with chronic graft-versus-host disease of the liver after an allogeneic bone marrow transplant. All of the patients presented with a total cholesterol in excess of 1000 mg/dl (25.9 mmol/l). At the time they were also noted to have pseudohyponatremia. Cholesterol appeared to be predominantly carried by lipoprotein X. Intrahepatic cholestasis leading to reflux of bile lipoproteins into the bloodstream and subsequent formation of protein X appears to be the mechanism underlying this phenomenon. Complications, including retinal cholesterol thromboembolism and cholesteroloma of the lung have been seen in the patient with the highest cholesterol levels. Severe hypercholesterolemia is an important, and likely more common than previously reported, long-term complication of allogeneic hematopoietic stem cell transplantation. It is important for clinicians to familiarize themselves with the diagnostic and therapeutic challenges this condition presents.

Use of human immunodeficiency virus-1 protease inhibitors is associated with atherogenic lipoprotein changes and endothelial dysfunction.

BACKGROUND: Human immunodeficiency virus protease inhibitors (HIV PIs) are associated with hyperlipidemia, hyperglycemia, and obesity; however, it is not known whether they increase risk of atherosclerotic vascular disease. The purposes of this study were to characterize the lipoprotein abnormalities associated with use of HIV PIs in individuals with HIV infection and to determine the pathophysiological significance of these changes by assessing their effect on endothelial dysfunction. METHODS AND RESULTS: This was a cross-sectional study of 37 adults with HIV-1 infection who were receiving antiretroviral therapy. Twenty-two were taking HIV PIs (group 1); 15 were not (group 2). Lipids and lipoproteins were measured by enzymatic techniques and nuclear magnetic resonance spectroscopic analysis. Flow-mediated vasodilation (FMD) of the brachial artery was measured by high-resolution ultrasound. Subjects in both groups were similar in regard to age, time since diagnosis of HIV infection, and CD4 cell count. Group 1 subjects had higher total cholesterol (5.68 versus 4.42 mmol/L, P=0.007) and triglyceride (4.43 versus 1.98 mmol/L, P=0.009) levels, characterized by elevated levels of IDL and VLDL. Subjects in group 1 had impaired FMD (2.6+/-4.6%), indicative of significant endothelial dysfunction. Group 2 subjects had normal FMD (8.1+/-6.7%, P=0.005). In group 1, chylomicron, VLDL, IDL, and HDL cholesterol levels predicted FMD. CONCLUSIONS: Use of HIV PIs is associated with atherogenic lipoprotein changes and endothelial dysfunction. Because these metabolic and vascular changes predispose to atherosclerosis, monitoring and treatment of dyslipidemia in patients taking these medications is warranted.

Purple grape juice improves endothelial function and reduces the susceptibility of LDL cholesterol to oxidation in patients with coronary artery disease.

BACKGROUND: In vitro, the flavonoid components of red wine and purple grape juice are powerful antioxidants that induce endothelium-dependent vasodilation of vascular rings derived from rat aortas and human coronary arteries. Although improved endothelial function and inhibition of LDL oxidation may be potential mechanisms by which red wine and flavonoids reduce cardiovascular risk, the in vivo effects of grape products on endothelial function and LDL oxidation have not been investigated. This study assessed the effects of ingesting purple grape juice on endothelial function and LDL susceptibility to oxidation in patients with coronary artery disease (CAD). METHODS AND RESULTS: Fifteen adults with angiographically documented CAD ingested 7.7+/-1.2 mL. kg(-1). d(-1) of purple grape juice for 14 days. Flow-mediated vasodilation (FMD) was measured using high-resolution brachial artery ultrasonography. Susceptibility of LDL particles to oxidation was determined from the rate of conjugated diene formation after exposure to copper chloride. At baseline, FMD was impaired (2.2+/-2. 9%). After ingestion of grape juice, FMD increased to 6.4+/-4.7% (P=0.003). In a linear regression model that included age, artery diameter, lipid values, and use of lipid-lowering and antioxidant therapies, the effect of grape juice on FMD remained significant (mean change 4.2+/-4.4%, P<0.001). After ingestion of grape juice, lag time increased by 34.5% (P=0.015). CONCLUSIONS: Short-term ingestion of purple grape juice improves FMD and reduces LDL susceptibility to oxidation in CAD patients. Improved endothelium-dependent vasodilation and prevention of LDL oxidation are potential mechanisms by which flavonoids in purple grape products may prevent cardiovascular events, independent of alcohol content.

Assessment of interlaboratory performance in external proficiency testing programs with a direct HDL-cholesterol assay.

Direct assays for the determination of HDL-cholesterol (HDL-C) have recently become available. The methods are precise, require small sample volume, and appear to be less affected by increased triglycerides than traditional precipitation methods. In this study, we describe the inter- and intralaboratory variability of the Boehringer Mannheim Corporation direct HDL-C assay and its performance in external proficiency testing surveys. A comparison study among three laboratories, using different analyzers and 85 serum specimens, showed a correlation coefficient (r) of 0.99. The direct HDL-C assay also showed good agreement with the ultracentrifugation-dextran sulfate-Mg2+ method (r = 0.98) and the Cholesterol Reference Method Laboratory Network-Designated Comparison Method (a = 0.98x + 4.75 mg/L, r = 0.98). Total error at medical decision levels ranged from -0.8% to +11.1%. Furthermore, this assay performed adequately in the College of American Pathologists and the ALERT surveys as well as the CDC Lipid Standardization Program and met all performance criteria of regulatory agencies.

Characterization of the peptide binding motif of a rhesus MHC class I molecule (Mamu-A*01) that binds an immunodominant CTL epitope from simian immunodeficiency virus.

The majority of immunogenic CTL epitopes bind to MHC class I molecules with high affinity. However, peptides longer or shorter than the optimal epitope rarely bind with high affinity. Therefore, identification of optimal CTL epitopes from pathogens may ultimately be critical for inducing strong CTL responses and developing epitope-based vaccines. The SIV-infected rhesus macaque is an excellent animal model for HIV infection of humans. Although a number of CTL epitopes have been mapped in SIV-infected rhesus macaques, the optimal epitopes have not been well defined, and their anchor residues are unknown. We have now defined the optimal SIV gag CTL epitope restricted by the rhesus MHC class I molecule Mamu-A*01 and defined a general peptide binding motif for this molecule that is characterized by a dominant position 3 anchor (proline). We used peptide elution and sequencing, peptide binding assays, and bulk and clonal CTL assays to demonstrate that the optimal Mamu-A*01-restricted SIV gag CTL epitope was CTPYDINQM(181-189). Mamu-A*01 is unique in that it is found at a high frequency in rhesus macaques, and all SIV-infected Mamu-A*01-positive rhesus macaques studied to date develop an immunodominant gag-specific CTL response restricted by this molecule. Identification of the optimal SIV gag CTL epitope will be critical for a variety of studies designed to induce CD8+ CTL responses specific for SIV in the rhesus macaque.

Comparison of NCEP performance specifications for triglycerides, HDL-, and LDL-cholesterol with operating specifications based on NCEP clinical and analytical goals.

The National Cholesterol Education Program (NCEP) performance specifications for methods that measure triglycerides, HDL-cholesterol, and LDL-cholesterol have been evaluated by deriving operating specifications from the NCEP analytical total error requirements and the clinical requirements for interpretation of the tests. We determined the maximum imprecision and inaccuracy that would be allowable to control routine methods with commonly used single and multirule quality-control procedures having 2 and 4 control measurements per run, and then compared these estimates with the NCEP guidelines. The NCEP imprecision specifications meet the operating imprecision necessary to assure meeting the NCEP clinical quality requirements for triglycerides and HDL-cholesterol but not for LDL-cholesterol. More importantly, the NCEP imprecision specifications are not adequate to assure meeting the NCEP analytical total error requirements for any of these three tests. Our findings indicate that the NCEP recommendations fail to adequately consider the quality-control requirements necessary to detect medically important systematic errors.

Optimized high-performance liquid chromatographic method for determination of lamotrigine in serum with concomitant determination of phenytoin, carbamazepine, and carbamazepine epoxide.

Lamotrigine (LG), phenytoin (PY), carbamazepine (CM), and carbamazepine epoxide (CE) are measured with an optimized procedure that uses thin sorbent extraction disks and a highly selective, sterically protected bonded silica high-performance liquid chromatography (HPLC) column. Routinely, serum (200 microliters at pH 6.8 with cyheptamide as internal standard) is applied to an Empore octyl (C8) solid-phase extraction disk to isolate the drugs. a water wash removes interferences, and the retained drugs are eluted with a small volume of solvent. The eluate is directly injected onto a Zorbax Stable Bond cyanopropyl HPLC column with quantification at 214 nm. Evaporation-concentration steps are unnecessary. Overall, for all drugs, between-run precision coefficients of variation (n = 16 each) ranged from 2.1% to 4.9% at concentrations from 0.75 to 20.5 mg/l; extraction recoveries fell within a range of 96% to 110% at concentrations of 2, 10, and 30 mg/l tested for each drug; the lowest limit of detection was 0.15 to 0.35 mg/l. The analytical response was linear for each drug > 80 mg/l (LG) and > 50 mg/l (PY, CM, and CE). Optimization graphs are presented to illustrate the rationale for selection of test parameters for a robust method. In addition, a comparison study between two commercial laboratories demonstrates accuracy problems associated with LG testing.

Optimized method for determination of gabapentin in serum by high-performance liquid chromatography.

The anticonvulsant drug gabapentin and its heptaneacetic acid analog-used here as an internal standard--are isolated from serum (pH 9) with an octyldecyl (C-18) solid-phase sorbent column. To enhance analytical detection, trinitrobenzene derivatives of these extracted compounds are prepared quickly within 10 min. To further improve chromatographic selectivity, the derivatives are concentrated on a thin C-18 solid-phase membrane and interferences are washed away. The retained purified derivatives are eluted from the membrane with a small volume of solvent and the eluate is directly injected onto an Ultrasphere C-18 high-performance liquid chromatography column with quantification at 340 nm. No evaporation-concentration steps are necessary. Recoveries (extraction) of gabapentin and the internal standard are 94.2 +/- 2.9% and 98 +/- 2.0%, respectively. Analytical responses are linear from lower limit of sensitivity of 0.05 mg/L up to at least 10 mg/L. Between-run coefficients of variation (CV) range from 2.3 to 2.9% through the concentration range 0.5-4.0 mg/L. To illustrate the rationale for selection of test parameters for a robust method, we present optimization graphs for these processes. Moreover, we discuss the advantage of the packed cartridge and membrane sorbens as companion extraction devices.

Use of particle-loaded membranes to extract steroids for high-performance liquid chromatographic analyses improved analyte stability and detection.

Cortisol, cortisone, corticosterone, prednisone and prednisolone are extracted from serum using the novel particle-loaded octyl (C8)-bonded silica in PTFE membrane. Extracts are directly injected, without further concentration, onto a narrow (2.0 mm) or conventional (4.6 mm) bore octyldecyl (C18) HPLC column. Method performance data demonstrate linearity from 0.4 microgram/dl (low limit of detection) up to at least 60 micrograms/dl. Extraction recoveries exceeded 85% and precision (between-run) R.S.D.s averaged < 5%. Interferences were minimal and selectivity was improved over conventional immunochemical steroid assays. When compared to large particle sorbents packed in columns or to traditional liquid-liquid extractions, the membrane extracted steroids in less time, used less reagent, and had smaller elution volumes, thereby obviating steroid instability/adsorption problems associated with traditional concentrating techniques required to improve analytical sensitivity.

Effects of tamoxifen on cardiovascular risk factors in postmenopausal women after 5 years of treatment.

BACKGROUND: Adjuvant tamoxifen therapy for breast cancer has been given for a period of several years. Cardiovascular diseases increased in incidence rapidly in women older than 60 years. Favorable changes in cardiovascular risk factors have been seen with 2 years of tamoxifen therapy, and lower rates of myocardial infarction and of hospitalization for heart disease have been observed in tamoxifen-treated women. PURPOSE: We sought to evaluate changes in risk factors for cardiovascular diseases in postmenopausal women after therapy with tamoxifen for 5 years. METHODS: Five years after their initial entry in a 2-year randomized, placebo-controlled toxicity study, we re-examined 62 of the original 140 disease-free, axillary node-negative postmenopausal breast cancer patients. These 62 patients were women available for study because they had not suffered major illness and had continued on either the tamoxifen or no-tamoxifen regimen to which they had been originally randomly assigned for the entire 5 years. Patient and control blood samples were analyzed for total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and subfractions, triglycerides, apolipoprotein AI, apolipoprotein B, lipoprotein(a), fibrinogen, glucose, and platelets. RESULTS: At base line for all measurements except atherogenic lipoprotein [lipoprotein(a)], the 30 long-term tamoxifen recipients and the 32 long-term no-tamoxifen recipients were not significantly different. After 5 years, levels of total serum cholesterol (P < .001), LDL cholesterol (P < .001), and lipoprotein(a) (P = .001) were significantly lower, and apolipoprotein AI levels were significantly higher (P < .001) in the tamoxifen-treated group compared with the no-tamoxifen group. Apolipoprotein B levels increased to a greater extent in the no-tamoxifen than in the tamoxifen group (P < .001). After 5 years, fibrinogen level decrease and triglyceride level increases in the tamoxifen group compared with the no-tamoxifen group were of borderline statistical significance and HDL cholesterol levels were not different in the two groups. CONCLUSION: Favorable changes in lipid, lipoprotein, and fibrinogen levels seen early in tamoxifen therapy in postmenopausal women persist with treatment of 5 years. IMPLICATIONS: The types and magnitude of changes in cardiovascular risk factors seen here with tamoxifen are similar to a certain extent with those seen with estrogen supplements. Further risk-factor and ethnic-group data are needed to estimate the magnitude of expected benefits of tamoxifen treatment on incidence of heart disease.

Lipid testing for the year 2000 and beyond.

Lipid testing has progressed from early measurements of total lipid by extraction and weighing to assess the fat content of the specimen. This nonspecific approach to lipid testing has been replaced in clinical laboratories by automated and quantitative procedures that avoid the extraction process. Instead, selective enzymes are utilized in reaction schemes to quantitate the individual lipid classes present in patient specimens. For example, cholesterol esterase and oxidase are used on a routine basis to measure total cholesterol in plasma and serum specimens. Similar use of other enzyme systems has permitted triglycerides and phospholipids to be measured by clinical laboratories. Lipid and lipoprotein measurements have advanced considerably from the early nonspecific extraction and gravimetric analysis schemes to the specific automated procedures that are commonly used today. However, as lipids and lipoproteins increased in their clinical usefulness as cardiovascular risk assessment tools, the search intensified for newer approaches to measure these entities more easily and more accurately. The influence of National Cholesterol Education Program has played a key role in highlighting the importance of lipids and lipoprotein analysis. Today, lipid testing is available outside the traditional laboratory environments - drugstores sell units that individuals can use at home to assess cholesterol levels. Lipid testing has come a long way, and we have only begun to experience some of the remarkable changes for the future.

Cholesterol--a model system to relate medical needs with analytical performance.

The evolution of cholesterol testing provides an example of a systematic approach that developed to relate the medical use of a laboratory test with the analytical performance requirements for that test. Laboratories today have the capability to perform cholesterol testing with the accuracy and precision necessary to meet medical needs. This statement can be made because (a) a standard diagnostic process has been established by the National Cholesterol Education Program; (b) an accuracy base is provided through a reference method that is readily available to manufacturers and laboratories; (c) the precision of analytical systems has been improved by manufacturers; (d) operating specifications for all such systems can be established, with statistical quality-control rules to ensure adequate within-run method performance; and (e) analytical performance is monitored by proficiency testing by using national quality requirements defined by CLIA '88 for acceptability. This cholesterol model provides a logical and scientific approach that should be applicable with other analytes to assure that the analytical performance of the laboratory test satisfies medical needs.

Correlations between scores on the Wechsler Intelligence Scale for Children-III and the General Purpose Abbreviated Battery of the Stanford-Binet IV.

Estimations of some relationships among scores on the "General Purpose Abbreviated Battery" of the Stanford-Binet: Fourth Edition and the Wechsler Intelligence Scale for Children-III were based on the responses of 14 boys and 18 girls enrolled in Grades 3, 4, and 5 and who took both tests. Of 13 Pearson correlations between the Binet IV composite score and the Wechsler subtest scores and IQs 12 were statistically significant (rs = .45 to .74). The new Wechsler scale appears to be a valid instrument for the 32 children (8-8 to 11-11) who were tested.

Application of the Empore solid-phase extraction membrane to the isolation of drugs from blood: II. Mexiletine and flecainide.

A stabilized therapeutic drug monitoring procedure incorporating the novel Empore solid-phase extraction membrane (SPEM) for isolation of the antiarrhythmic drugs mexiletine (MEX) and flecainide (FLEC) from serum is described. Routinely, serum (0.5 ml), adjusted to pH 4.5, is passed through an octyl (C8) SPEM to extract the drugs. A methanol:water wash follows to remove proteins and interferences. MEX and FLEC are eluted from the membrane with mobile phase and an aliquot is injected directly onto a Zorbax cyanopropyl (CN) high-performance liquid chromatographic column with detection at 214 nm. Evaporating/concentrating techniques that can adversely influence the stability of the volatile MEX are unnecessary. Recovery for both drugs exceeds 90% and the assay is linear from 0.05 mg/L up to at least 6.0 mg/L for MEX and from 0.05 mg/L up to at least 3.0 mg/L for FLEC. Precision (between-run) coefficients of variation range from 2.3 to 3.0% (0.49-1.97 mg/L) for MEX and 3.7 to 5.9% (0.240-0.992 mg/L) for FLEC. Interferences are minimal. When we compared performance of the Empore SPEM and large-particle solid-phase sorbents packed in cartridges, we observed greater capacity per gram of sorbent and smaller elution volume with the membrane. Most important, concentrating steps that adversely affect the stability of MEX are avoided with the SPEM.

Effects of tamoxifen on cardiovascular risk factors in postmenopausal women.

OBJECTIVE: To determine the effects of tamoxifen on risk factors for cardiovascular disease in disease-free postmenopausal women. DESIGN: Double-blind, placebo-controlled, randomized 2-year clinical trial. SETTING: University health sciences center. PATIENTS: Clinically postmenopausal women (140) with a diagnosis of axillary node-negative breast cancer, who were disease-free by laboratory and clinical evaluations. MEASUREMENTS: Levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, apolipoprotein A-I, apolipoprotein B, glucose, weight, blood pressure, and reported exercise and work activity were measured. MAIN RESULTS: Postmenopausal women receiving tamoxifen were evaluated at 3- or 6-month intervals during a 2-year assessment period and showed a mean decrease of 12% in total cholesterol levels (at 24 months -0.672 mmol/L; 95% CI, -0.839 to -0.505 mmol/L) and a mean decrease of 20% in calculated low-density lipoprotein (LDL) cholesterol levels (at 24 months, -0.725 mmol/L; 95% CI, -0.868 to -0.583 mmol/L) (P less than 0.001). Women with greater baseline cholesterol levels had greater decreases with tamoxifen treatment. Levels of HDL cholesterol decreased in patients treated with tamoxifen, but this decrease was only statistically significant at one of five measurement times. Apolipoprotein A-I levels increased significantly at the two time points at which it was measured (P = 0.02), and apolipoprotein B levels decreased significantly at these times (P less than 0.01) in patients treated with tamoxifen. Plasma glucose levels, reported exercise and work activity, reported smoking, weight, and systolic and diastolic blood pressures did not change with treatment. CONCLUSION: During 2 years of treatment, tamoxifen showed generally favorable effects on the lipid and lipoprotein profile of treated postmenopausal women. These effects may partially explain the decrease in adverse events and in mortality related to coronary heart disease seen in patients receiving adjuvant tamoxifen treatment.

Cholesterol operational process specifications for assuring the quality required by CLIA proficiency testing.

Current U.S. governmental regulations and requirements for the quality of laboratory tests do not provide a consistent form, comparable numbers, or practical specifications for the routine operation of laboratory testing processes. For cholesterol, as an example, the Health Care Financing Administration provides an analytical performance criterion for proficiency testing to enforce the Clinical Laboratory Improvement Act (CLIA), whereas the U.S. National Cholesterol Education Program (NCEP) provides clinical guidelines for test interpretation, as well as analytical goals for imprecision and inaccuracy. Routine operating process specifications for imprecision, inaccuracy, and quality control can be derived from the analytical and clinical requirements for quality. Use of an analytical "total error" model and a clinical "decision interval" model provides logically consistent and numerically comparable specifications. Studies with these coherent models indicate that a cholesterol testing process properly planned to satisfy the CLIA analytical requirement will also satisfy the NCEP clinical requirement. To provide 90% assurance of detecting systematic shifts of a magnitude that would cause the CLIA analytical requirement to be exceeded, the operational specifications for a cholesterol testing process are an allowable CV of less than or equal to 2%, an allowable bias of less than or equal to 1%, and a control procedure with two measurements per run interpreted by 1(3)s, 1(2.5)s, or 1(3)s/2(2)s/R4s control rules.

Application of a novel form of solid-phase sorbent (Empore membrane) to the isolation of tricyclic antidepressant drugs from blood.

We employed a new form of solid-phase material, the Empore octyl (C8) extraction membrane (SPEM), for the efficient extraction of tricyclic drugs from patients' serum specimens. Both extraction and companion high-performance liquid chromatographic (HPLC) assay of doxepin (DOX), desmethyldoxepin (DDOX), imipramine (IMI), desmethylimipramine (DESI), amitriptyline (AMI), nortriptyline (NOR), clomipramine (CLO), and desmethylchlomipramine (DCLO) are presented here. Routinely, serum (1.0 mL or less) adjusted to pH 5.5 with phosphate buffer is passed through the SPEM secured in a MF-1 microfilter unit. Proteins and potential interferences retained on SPEM are removed with an acetonitrile-water wash. The tricyclic drugs are eluted with HPLC mobile phase and the eluate is injected directly on a Zorbax cyanopropyl (CN) HPLC column, thereby avoiding time consuming evaporation-concentration steps that can affect drug stability. Recovery for all drugs exceeds 90% and analytical responses are linear from a lower limit of sensitivity of 8 micrograms/L up to at least 1000 micrograms/L. Between-run coefficients of variation (CV) range from 2.9 to 8.3% through the concentration range of 75 to 300 micrograms/L. Performance characteristics of the SPEM are compared to those of conventional large particle silica- and polymeric-based sorbents. Within the requirements of this assay, the SPEM extraction requires less sample volume and reduces elution and solvent volumes.

Synergistic and antagonistic effects of combinations of cyclosporine A and its metabolites on inhibition of phytohemagglutinin-induced lymphocyte transformation in vitro.

Cyclosporine A (CsA) and purified CsA metabolites were tested alone and in combination in cell culture to determine their effects on phytohemagglutinin (PHA)-induced lymphocyte proliferation. CsA was significantly more inhibitory than its metabolites at all concentrations tested (0-1000 ng/mL). CsA exerted maximum inhibition (70% decrease in [methyl-3H]thymidine incorporation) at concentrations of 300 ng/mL or greater; metabolites M1, M17, and M21 depressed the response 46, 39, and 23%, respectively, at 300 ng/mL. Metabolites M8, M18, M26, M25, M13, and M203-218 were non-inhibitory. When combinations of M17 and CsA were tested for the effects on PHA-induced lymphocyte transformation, a synergistic effect occurred at combinations of low concentrations of M17 and CsA and an antagonistic effect at the higher concentrations. Of the 49 combinations of CsA and M17 tested, 30 were antagonistic, 16 synergistic and 3 undecided (approaching addition). When 49 combinations of CsA and the non-immunosuppressive metabolite M8 were tested, 29 of the 49 combinations were synergistic, 17 antagonistic, 1 additive and 2 undecided (approaching addition). Of the 29 synergistic combinations, 14 were strongly synergistic. The importance of the interaction of CsA and metabolites to the immunopharmacology of CsA therapy is discussed.

Application of the Empore solid-phase extraction membrane to the isolation of drugs from blood. I. Amiodarone and desethylamiodarone.

We describe the use of a new form of solid-phase material, the Empore solid-phase extraction membrane (SPEM), for therapeutic drug monitoring. We evaluated the new extraction procedure with the companion high-performance liquid chromatographic (HPLC) method for the antiarrhythmic drug amiodarone and its metabolite, desethylamiodarone, in patients' serum. Acidified serum (250 microliters) was passed through an octyl (C8) SPEM secured in an MF-1 microfilter unit. Serum proteins and potential interferences were removed with an acetonitrile:water wash, and the retained drugs eluted with HPLC mobile phase. This eluate was injected directly onto the analytical column. Both drugs averaged 85% recovery with a linear response from a lower limit of detection at 0.05 mg/L up to 6 mg/L, and between-run precision coefficients of variation ranging from 3.1 to 6.4% over the concentration range of 0.5-3.0 mg/L. We observed significant advantages of the novel SPEM over conventional liquid-liquid or large-particle size solid-phase sorbents packed in cartridges. Minimal amounts of solvents were required, elution volume was smaller, time-consuming evaporating/concentrating steps that can influence drug stability were avoided, and little throw-away material was generated. Only the small membrane was discarded.