The C-3 epimer of 25-hydroxyvitamin D(3) is present in adult serum.

CONTEXT: Epimers have identical molecular structure but differ in stereochemical configuration. It is widely believed that the C-3 epimer of 25-hydroxyvitamin D(3) [3-epi-25(OH)D(3)] is found only in neonates. However, this epimer was recently detected in a limited number of adults. The physiological importance of 3-epi-25(OH)D(3) is uncertain but might affect 25-hydroxyvitamin D test results and thereby reliability of the 25-hydroxyvitamin D(3) [25(OH)D(3)] measurement. OBJECTIVE: This project describes development of a highly sensitive method for 3-epi-25(OH)D(3) measurement and establishes the prevalence of this epimer in adult clinical serum specimens. DESIGN, SETTING, PARTICIPANTS, AND MAIN OUTCOME MEASURE: Serum 25(OH)D(3), 3-epi-25(OH)D(3), and 25(OH)D(2) concentrations were determined in a cohort of patients (n = 214; age neonate to 80+ yr). High-performance liquid chromatography with ultraviolet detection and high-performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization equipped with cyanopropyl analytical columns were used to baseline separate and quantitate 25(OH)D(3), 3 epi-25(OH)D(3), and 25(OH)D(2). RESULTS: The C-3 epimer was detected in 212 of 214 (99%) of samples. Concentrations ranged from 1 to 93 ng/ml for 25(OH)D(3) and 0.1 to 23.7 ng/ml for 3-epi-25(OH)D(3). The relative amounts of epimer to 25(OH)D(3) ranged from 0 to 25.5% (mean 4.75%). The epimer amount increased as 25(OH)D(3) increased in a nonlinear mode. In sera with approximately the same 25(OH)D(3) concentration, the ratio of epimer to 25(OH)D(3) varied, e.g. at 25(OH)D(3) values of 20-22 ng/ml, the ratio varied from 2-8.5%. CONCLUSION: 3-Epi-25(OH)D(3) is present in the majority of human serum specimens. Although this concentration is generally low, further work must investigate the impact of 3-epi-25(OH)D(3) on the various 25-hydroxyvitamin D assays and ultimately what information, if any, C-3 epimer measurement can provide clinically.

Low vitamin D status despite abundant sun exposure.

CONTEXT: Lack of sun exposure is widely accepted as the primary cause of epidemic low vitamin D status worldwide. However, some individuals with seemingly adequate UV exposure have been reported to have low serum 25-hydroxyvitamin D [25(OH)D] concentration, results that might have been confounded by imprecision of the assays used. OBJECTIVE: The aim was to document the 25(OH)D status of healthy individuals with habitually high sun exposure. SETTING: This study was conducted in a convenience sample of adults in Honolulu, Hawaii (latitude 21 degrees ). PARTICIPANTS: The study population consisted of 93 adults (30 women and 63 men) with a mean (sem) age and body mass index of 24.0 yr (0.7) and 23.6 kg/m(2) (0.4), respectively. Their self-reported sun exposure was 28.9 (1.5) h/wk, yielding a calculated sun exposure index of 11.1 (0.7). MAIN OUTCOME MEASURES: Serum 25(OH)D concentration was measured using a precise HPLC assay. Low vitamin D status was defined as a circulating 25(OH)D concentration less than 30 ng/ml. RESULTS: Mean serum 25(OH)D concentration was 31.6 ng/ml. Using a cutpoint of 30 ng/ml, 51% of this population had low vitamin D status. The highest 25(OH)D concentration was 62 ng/ml. CONCLUSIONS: These data suggest that variable responsiveness to UVB radiation is evident among individuals, causing some to have low vitamin D status despite abundant sun exposure. In addition, because the maximal 25(OH)D concentration produced by natural UV exposure appears to be approximately 60 ng/ml, it seems prudent to use this value as an upper limit when prescribing vitamin D supplementation.

Therapeutic monitoring of tacrolimus concentrations in blood: semi-automated extraction and liquid chromatography-electrospray ionization mass spectrometry.

The authors describe a highly selective liquid chromatographic/mass spectrometric (LC/MS) method for measurement of the immunosuppressive drug tacrolimus. A protein-free supernatant of a patient's whole-blood sample (500 microL) is applied to an Empore styrene-divinylbenzene (SDB-XC) disk cartridge (Varian Sample Preparation Products; Harbor City, CA) to isolate tacrolimus and the internal standard, ascomycin. The entire extraction is automated on the Gilson ASPEC XL4 (Gilson; Middleton, WI). Separation takes place on an octyldecyl (C18) high-performance liquid chromatographic (HPLC) column maintained at 75 degrees C with a simple mobile phase of acetonitrile/water (90/10, v/v). An atmospheric pressure ionization (API)-electrospray ionization(ESI)-mass spectrometer (MS) is used for analysis. Detection is by selected ion monitoring (SIM) of the positively charged sodium adduct of tacrolimus and ascomycin, m/z 826.5 and 814.5, respectively. Collision-induced dissociation (CID) fragment ions of tacrolimus and ascomycin (m/z 616.4 and 604.4, respectively) are also monitored to enhance overall selectivity. Total run time is 1 minute per injection. A plot of chromatographic peak height ratio of m/z 826.5 to m/z 814.5 vs tacrolimus blood concentration is linear from the lowest limit of quantitation (0.3 microg/L) to at least 120.0 microg/L. Metabolites of tacrolimus and other immunosuppressive and commonly prescribed drugs do not interfere. Analytical recovery is 94% to 113% over a range of 4.4 to 41.0 microg/L. Between-run precision coefficients of variation (CV) are 3.6% to 4.2% over a range of 8.1 to 32.4 microg/L. Comparison data (n = 156) of the LC/MS method versus a commercial immunoassay (Abbott Tacrolimus IMx MEIA II) (Abbott; Chicago, IL) demonstrated that the tacrolimus concentration as measured by LC/MS = (0.912 x MEIA concentration) - 0.049; r2 = 0.968. This validated LC/MS method demonstrates significant advantages over conventional immunoassays and improves on the lower limit of detection, sensitivity, accuracy, and range of linearity. In the authors' laboratory, this method is approximately 60% less costly to perform than the most commonly implemented immunoassay. Together, the authors' daily clinical experience during 1 year and participation in a proficiency testing program has demonstrated that the method is robust and suitable for routine therapeutic monitoring of tacrolimus.

The effects of ketoconazole on the pharmacokinetics of cyclosporine A in cats.

OBJECTIVE: To determine the effects of ketoconazole (KC) on the pharmacokinetics of cyclosporine A (CsA) elimination in cats. STUDY DESIGN: Research study and prospective clinical trial. ANIMALS: Five healthy adult cats (pharmacokinetic studies) and 6 client-owned cats with chronic renal failure. METHODS: Blood CsA concentrations were measured after CsA (4 mg/kg i.v.) administration with or without concurrent oral KC (10 mg/kg). Subsequently, a combined CsA-KC immunosuppressive regimen was used in cats after kidney transplantation. Blood CsA concentrations were measured using high performance liquid chromatography. CsA elimination was analyzed using a computerized pharmacokinetics program. RESULTS: KC increased blood CsA concentrations 1.8-fold and 2.2-fold at 12 and 24 hours after CsA administration. KC significantly decreased the mean systemic CsA clearance from 2.73 mL/min/kg to 1.22 mL/min/kg resulting in an increase in the terminal phase half-life from 10.7 to 22.2 hours. The volume of distribution of steady-state of CsA was unaffected by KC. In a series of clinical feline kidney transplant patients, a once-a-day CsA-KC regimen was able to be used in most of the cats and was effective for prevention of allograft rejection in all of these cats. CONCLUSION AND CLINICAL RELEVANCE: KC is an effective adjunct treatment for immunosuppression in feline kidney transplant patients. KC suppresses CsA elimination, which reduces the need for CsA and allows once daily administration of CsA.

Therapeutic monitoring of topiramate: evaluation of the saturable distribution between erythrocytes and plasma of whole blood using an optimized high-pressure liquid chromatography method.

Topiramate (TPM) reportedly binds in a saturable manner to erythrocytes but minimally to plasma proteins. Two studies were performed to evaluate this distribution phenomenon. In all studies, TPM was measured with a newly developed, optimized procedure that uses octyldecyl (C-18) solid phase sorbents disks/packed cartridges and a DB-1 methylsilicone capillary gas chromatography (GC) column. Between-run precision coefficients of variation (CVs) (n = 16) ranged from 3.6%-5.6% at concentrations from 3.0 to 15 microg/mL, with low limit of detection of 0.2 to 0.3 microg/mL. For the distribution studies, drug-free whole-blood specimens from five healthy adult volunteers were supplemented with TPM and used to test the influence of TPM concentration and HCT differences on the plasma/blood (P/B) distribution ratio of TPM. In study A, TPM concentration was varied (1-15 microg/mL) and HCT remained constant (40% +/- 5%). In study B, TPM (3 microg/mL) was added to blood specimens comprising a range of HCT values (20%-40%). Study A results were: mean TPM P/B ratios: 0%, 14.2% +/- 5%, 44.2% +/- 4%, 76% +/- 5.5% at 1, 3, 5, 15 microg/mL, respectively. Data between each group were statistically different (p < 0.001). Study B results were: mean TPM P/B ratio: 17.3% +/- 7.3%, 27.5% +/- 10.1%, 39.8% +/- 8% and 56.1% +/- 8.8% at HCT values of 40%, 32%, 26.5%, 20%, respectively. The TPM P/B ratio was significantly inversely correlated to HCT (r = -905, p < 0.001). TPM P/B partitioning was not temperature-dependent. Researchers concluded that the saturable binding of TPM to RBC is significant and is correlated to HCT. As a result, TPM in the plasma fraction of whole blood will increase when HCT decreases and as total TPM concentration in whole blood increases.

Comparison of high performance liquid chromatography and immunoassay methods for measurement of cyclosporine A blood concentrations after feline kidney transplantation.

OBJECTIVE: To compare two methods of whole blood cyclosporine A (CsA) measurement in cats. STUDY DESIGN: Whole blood samples were analyzed for CsA concentrations with use of high performance liquid chromatography (HPLC) and monoclonal immunoassay methods. ANIMALS: Blood (n = 36 samples) was obtained from six cats after renal transplantation. METHODS: Results were compared by linear regression analysis using both pooled and individual patient data. Eight samples were off-scale on the immunoassay and were excluded. RESULTS: There was significant correlation between CsA measured using HPLC and immunoassay methods (P < .001; r = .942; r2 = .887). However, individuals varied nonrandomly from the mean pooled patient data. Correlation between the assay methods was higher for individual patients using data only from that specific individual (mean r value = .976; r2 = .955). Clinical utility of the immunoassay (ie, results would prompt an appropriate CsA dosage adjustment) was good when based on individually derived conversion factors (27 of 28 [96.5%] of decision events). CONCLUSION: HPLC is superior for measurement of blood CsA concentrations in cats after kidney transplantation. However, an immunoassay may provide reliable information for CsA management if a comparative database (HPLC v immunoassay) has been previously determined in a specific patient. CLINICAL RELEVANCE: Locally available monitoring of CsA by immunoassay in cats may provide significant advantages when shipping of blood samples to distant locations is required to obtain analysis by HPLC. These advantages may include cost and timeliness of results in circumstances where daily blood CsA concentrations may be desired, such as when managing an acute rejection reaction.

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.

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.

Lamotrigine pharmacokinetics in patients receiving felbamate.

Drug interactions can significantly complicate the management of patients receiving multiple medications. It is essential therefore that potential pharmcokinetic interactions be evaluated as new antiepileptic medications are introduced. Lamotrigine (LTG) is a recently marketed medication whose pharmacokinetics are significantly influenced by concomitant drugs. Felbamate (FBM), another relatively new antiepileptic agent has been associated with multiple interactions including both enzyme induction and inhibition. The purpose of the present pilot study was to evaluate potential differences in lamotrigine kinetics in six patients concomitantly receiving FBM compared to five patients receiving lamotrigine as monotherapy. There was no statistically significant differences in either apparent LTG oral clearance (0.026 +/- 0.005 vs. 0.024 +/- 0.01 l/kg per h, respectively), or in mean elimination half-life (33.7 +/- 7.5 vs. 40.2 +/- 15.05 h, respectively). Oral clearance values in our patients are also consistent with data reported previously in the literature. Data from this pilot study suggest that a marked effect of FBM upon lamotrigine pharmacokinetics is unlikely.

Effect of a high-protein meal on gabapentin pharmacokinetics.

The anticonvulsant gabapentin is transported across biological membranes via the L-amino acid transport system (System-L). Absorption of gabapentin is saturable, and in-vitro data have previously demonstrated that both L-leucine and L-phenylalanine may compete with the intestinal transport of gabapentin. The purpose of this study therefore was to determine whether a high-protein meal would interfere with gabapentin absorption. Ten healthy volunteers received in a randomized, cross-over design, a single 600-mg dose of gabapentin in the fasting state and after a high-protein meal consisting of 80 gm total protein (4.1 g phenylalanine, 8.2 g leucine and 4.2 g isoleucine), 52 g carbohydrate, and 9 g fat. Plasma gabapentin concentrations were measured by HPLC at baseline, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 30 h. Calculated pharmacokinetic parameters included Cmax' Tmax' AUC and T1/2. In addition, a pharmacodynamic assessment (using visual analog scales) of gabapentin-related adverse effects was performed at 2 h post drug ingestion and was compared between study phases. Statistical analysis included Student's t-test for paired data, with significance assigned at P < 0.05. Cmax was significantly increased by 36% (3.87 +/- 1.15 vs 5.28 +/- .97 micrograms/ml, P = 0.002), and Tmax tended to be shorter (3.9 +/- 1.8 vs 2.8 +/- .35 h, P = 0.10), after the high-protein meal. Although AUC was increased by 11%, this did not achieve statistical significance. Despite significantly higher plasma concentrations at 2 h, subjects reported significantly fewer adverse effects after the high-protein meal. Potential mechanisms to explain these unexpected findings may be that the large amino acid load delivered with the high-protein meal enhanced gabapentin absorption via trans-stimulation, the process by which acutely increased intestinal luminal amino acid concentrations result in an acute up regulation in System-L activity. Conversely, the decrease in perceived adverse CNS effects of gabapentin following the high-protein meal may reflect CNS competition for System-L transport.

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.

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.

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.

Concentrations of cyclosporin A and its metabolites in human tissues postmortem.

We report concentrations and distribution of cyclosporine A (CsA) and individual metabolites associated with various organ tissues and whole-blood specimens collected at autopsy from seven transplant patients who received CsA therapy. Solid-phase extraction (SPE) and specific high-performance liquid chromatographic (HPLC) procedures were used to separate and quantitate the cyclosporines. Patterns of deposition were unique for the various tissue types. Metabolites M17, M1, M18, and M8 (in addition to CsA) were the principal compounds detected in significant quantities. On a per weight basis, the sum concentration of CsA and metabolites in organ tissues was up to 53 times greater than in companion whole-blood specimens. Metabolite M17 prevailed in most tissues, except in fat and pancreas, where CsA was predominant. Overall, pancreas specimens contained a greater concentration of cyclosporines (per kilogram of tissue), followed consecutively by spleen, liver, fat, kidney, lung, bone marrow, heart, and whole blood. No CsA-related compounds were detected in brain or spinal cord tissue.

Three commercial polyclonal immunoassays for cyclosporine in whole blood compared: 1. Results with patients' specimens.

We assessed the performance of three commercially available polyclonal immunoassays for apparent cyclosporine in 120 whole-blood specimens collected from transplant recipients just before their next dose of cyclosporine (CsA). The assays were (a) Abbott's TDx fluorescent polarization immunoassay for CsA and its metabolites in whole blood; (b) the Sandoz radioimmunoassay (RIA); and (c) Incstar's Cyclo-Trac RIA. Mean respective CVs were 3.8%, 9.3%, and 24.3%. Analytical recovery was nearly 100% for concentrations up to 1000 micrograms/L for Incstar and up to 1500 micrograms/L for Abbott and Sandoz; linearity was compromised at greater concentrations. We also quantified the parent CsA concentrations by HPLC. Moreover, to follow day-to-day fluctuations in patients' "cyclosporine" concentrations with each method and to assess the impact these differences have on interpretation of the analytical results, we assayed serial specimens from six post-transplant patients. These showed significantly dissimilar, but parallel, results among the methods for any single sample. Occasionally, however, a result would not fit the established trend. Biases observed among the assays can be explained in part by the nonspecific antisera cross-reacting with CsA metabolites. Most important, we demonstrate that patients' results are not reliably interchangeable among the methods.

Three commercial polyclonal immunoassays for cyclosporine in whole blood compared: 2. Cross-reactivity of the antisera with cyclosporine metabolites.

We demonstrate the diverse selectivity of three commercial polyclonal "cyclosporine" immunoassays for cyclosporin (CsA) metabolites by comparing analytical responses of nine metabolites added to drug-free whole-blood specimens (range 0 to 2000 micrograms/L) and assayed by the Abbott TDx fluorescence polarization immunoassay (FPIA), the Incstar Cyclo-Trac radioimmunoassay (RIA), and the Sandoz RIA. Cross-reactivity--defined as the relative response (slope of regression line) of metabolite/parent CsA over the assay's linear range of concentrations--differed for each metabolite among the three assays. Overall, Abbott's antiserum exhibited the greatest affinity for the metabolites, the Sandoz antiserum the least. Ranges of cross-reactivity for the metabolites over all three assays were M1 (14-44%), M8 (9-20%), M13 (13-26%), M17 (50-116%), M18 (17-79%), M21 (4-54%), M25 (less than 1-52%), M26 (less than 1-29%), and M203-218 (7-51%). The specificities of the Abbott, Incstar, and Sandoz polyclonal assays thus differ significantly, and this brings into question the practical utility of comparing data generated for patients' specimens by different procedures.