Aliquots Of Plasma Research Articles

A simple and sensitive assay for the quantitative analysis of paclitaxel and metabolites in human plasma using liquid chromatography/tandem mass spectrometry

A sensitive and specific LC-MS/MS assay for the determination of paclitaxel and its 3'p- and 6-alpha-hydroxy metabolites is presented. A 200 microL plasma aliquot was spiked with a 13C6-labeled paclitaxel internal standard and extracted with 1.0 mL tert-butylmethylether. Dried extracts were reconstituted in 0.1 M ammonium acetate-acetonitrile (1:1, v/v) and 25 microL volumes were injected onto the HPLC system. Separation was performed on a 150 x 2.1 mm C18 column using an alkaline eluent (10 mm ammonium hydroxide-methanol, 30:70, v/v). Detection was performed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range for paclitaxel from 0.25 to 1000 ng/mL and metabolites from 0.25 to 100 ng/mL using 200 microL human plasma samples. Validation results demonstrate that paclitaxel and metabolite concentrations can be accurately and precisely quantified in human plasma. This assay is now used to support clinical pharmacologic studies with paclitaxel.

266 ANTIOXIDANT CAPACITY OF BOAR SEMINAL PLASMA

It has been established that antioxidants in seminal plasma play an important role in protecting the spermatozoa against oxidative stress-induced damage. This study was conducted to measure the total antioxidant capacity (TAC) of boar seminal plasma. Fifty-four ejaculates were collected from 17 mature boars of proven fertility by the gloved-hand technique. Ejaculates were collected separately in different fractions (pre-sperm, sperm-rich, and post-sperm) according to their macroscopic (color) characteristics. After centrifugation (2400g for 3 min), the sperm pellet was discarded; the supernatant was recentrifuged and filtered through a 10-μm nylon mesh filter to remove debris or clumped spermatozoa. The seminal plasma was frozen at -20°C until further use. After thawing at room temperature, seminal plasma aliquots of 5 μL were immediately assessed for total antioxidant capacity. TAC was measured using the ABTS/H2O2/HRP decoloration method (Cano A et al. 2000 Redox Report 5, 365–370) which allows differentiation between hydrophilic and lipophilic antioxidant activity capacity. TAC units were expressed as micromolar (μM) Trolox equivalents. Data were analyzed using ANOVA. Only the hydrophilic activity was measurable, with the lipophilic activity being undetected. The overall TAC of seminal samples (mean ± SEM) was 1623.7 ± 56.28 μM, ranging from 674 to 2428 μM. Different TACs were observed among males (P < 0.05) and between ejaculates of the same male (P < 0.05). Ejaculate fraction had a significant effect (P < 0.001) on the TAC levels. The post-sperm fraction had a significantly lower TAC level (1104.09 ± 57.66 μM) than the pre-sperm and sperm-rich fractions (1611.95 ± 153.68 μM and 1356.136 ± 72.47 μM, respectively, P < 0.001). In conclusion, hydrophilic antioxidant activity represented the main contribution to the TAC in boar seminal plasma, showing differences among males, between ejaculates of the same male, and also between the different ejaculate fractions. This work was supported by CICYT (AGF98-0533; AGL01-0471) and INIA (RZ01-019).

A novel liquid chromatography/tandem mass spectrometry based depletion method for measuring red blood cell partitioning of pharmaceutical compounds in drug discovery

A novel liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based depletion method for measuring compound partitioning between human plasma and red blood cells (RBC) in a drug discovery environment is presented. Conventionally, RBC partitioning is determined by separate measurements of drug concentrations in equilibrating plasma and whole blood or RBC using separate standards prepared in their respective matrices, i.e., in plasma and whole blood or RBC lysates. The process is very tedious, labor-intensive, and difficult to automate. In addition, interferences from the heme and other highly abundant cellular composites make the measurement of the drug concentration in whole blood or RBC inevitably variable even with a highly specific LC/MS/MS method. Therefore, there is an imminent need to develop a straightforward and fast method to assess the partitioning of drug-like compounds in RBC. This work describes an LC/MS/MS-based depletion assay that measures the compound concentration in plasma that has been equilibrating with RBC. Compounds were spiked into fresh human whole blood and plasma respectively to a final concentration of 500 nM. Both the spiked whole blood and plasma control were incubated at 37 degrees C for up to 60 min. During the time course, aliquots of plasma and whole blood from both incubation mixtures were sampled at 10 and 60 min. The whole blood samples were centrifuged to yield the plasma. The plasma samples from both incubations were extracted using a protein precipitation method, and analyzed using LC/MS/MS under the multiple-reaction monitoring (MRM) mode. The RBC partitioning ratio was calculated using the analyte peak area responses of the plasma samples through an equation deduced in this work. The method was first tested using two commercial compounds, phenoprobamate and acetazolamide, to determine the optimal incubation conditions and the concentration dependency of the assay. The assay reproducibility was also assessed by three inter-day assays for phenoprobamate. This method was further evaluated using 20 commercial compounds of different classes with a wide range of RBC partitioning coefficients and the results were compared with those reported in the literature. Excellent correlation (R2=0.9396) was found between the measured and literature values. In addition, several proprietary compounds were assayed using both the new and traditional methods and the measured partitioning ratios from the two methods are equivalent. The experiments in this work demonstrate that the LC/MS/MS-based depletion method can provide direct and accurate measurement of RBC partitioning for compounds in drug discovery.

Quantitative analysis of docetaxel in human plasma using liquid chromatography coupled with tandem mass spectrometry

An assay for the quantitative determination of docetaxel in human plasma is described. Docetaxel was extracted from the matrix using liquid-liquid extraction with ter-butylmethylether, followed by high-performance liquid chromatographic analysis using an alkaline eluent. Paclitaxel was used as internal standard. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. The validated range for docetaxel was from 0.25--1000 ng/mL using 200 microL plasma aliquots. The method requires only a limited volume (200 microL) of human plasma and the method can be applied in studies requiring a low lower limit of quantitation of 0.25 ng/mL. The assay was applied successfully in several clinical and pharmacological studies with docetaxel.

Effect of protease treatment on plasma infectivity in variant Creutzfeldt‐Jakob disease mice

The emergence of variant Creutzfeldt-Jakob disease (vCJD) and of a probable transmission of the disease through blood transfusion from a presymptomatic case has underlined the need for a reliable, sensitive, and specific screening test. This study was initiated to explain why attempts to identify protease-resistant prion protein (PrPres) following treatment with proteinase K (PK) in blood or blood components have so far failed. RIII mice were inoculated intracerebrally (i.c.) with vCJD agent. As soon as some mice became ill, blood from all mice was collected, pooled, and separated into components. Aliquots of plasma were treated with either 100 and 500 microg per mL PK or left untreated. Samples were analyzed for total protein and for PrPres by Western blot with 6H4 antibodies. Infectivity in PK-treated and untreated samples was bioassayed by i.c. inoculation into healthy mice. Estimated infectivity in untreated control plasma was 20.6 IU per mL. Treatment of plasma with 100 or 500 microg per mL PK resulted in estimated infectivity levels of 8.4 and 5.2 IU per mL, respectively. Coomassie staining revealed substantial changes in the protein profile after PK treatment, with massive degradation of proteins at 500 microg per mL PK. No PrPres was detected in plasma samples by Western blotting. Infectivity in plasma of vCJD-infected mice showed a trend toward reduction following enzymatic treatment with increasing doses of PK, possibly because of activity against proteolysis-sensitive isoforms of abnormal prion protein. It is concluded that the use of PK in protocols for the detection of PrPres may decrease the sensitivity of blood-based assays.

Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and absence of their sulfated counterparts in rodent brain

A new sample preparation method coupled to GC-MS analysis was developed and validated for quantification of sulfate esters of pregnenolone (PREG-S) and dehydroepiandrosterone (DHEA-S) in rat brain. Using a solid-phase extraction recycling protocol, the results show that little or no PREG-S and DHEA-S (<1 pmol/g) is present in rat and mouse brain. These data are in agreement with studies in which steroid sulfates were analyzed without deconjugation. We suggest that the discrepancies between analyses with and without deconjugation are caused by internal contamination of brain extract fractions, supposed to contain steroid sulfates, by lipoidal forms of PREG and DHEA (L-PREG and L-DHEA, respectively). These derivatives can be acylated very efficiently with heptafluorobutyric anhydride and triethylamine, and their levels in rodent brain (approximately 1 nmol/g) are much higher than those of their unconjugated counterparts. They are distinct from fatty acid esters, and preliminary data do not favor structures such as sulfolipids or sterol peroxides. Noncovalent interactions between steroids and proteolipidic elements, such as lipoproteins, could account for some experimental data. Given their abundance in rodent brain, the structural characterization and biological functions of L-PREG and L-DHEA in the central nervous system merit considerable attention.

A simple and sensitive assay for the quantitative analysis of paclitaxel in human and mouse plasma and brain tumor tissue using coupled liquid chromatography and tandem mass spectrometry

The development and validation of an assay for the determination of paclitaxel in human plasma, human brain tumor tissue, mouse plasma and mouse brain tumor tissue is described. Paclitaxel was extracted from the matrices using liquid-liquid extraction with tert-butyl methyl ether, followed by chromatographic analysis using an alkaline eluent. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicate that calibration standards in human plasma can be used to quantify paclitaxel in all tested matrices. In human samples, the validated range for paclitaxel was from 0.25-1000 ng ml(-1) using 200 microl plasma aliquots and from 5 to 5000 ng g(-1) using 50 microl tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma). In mice, the ranges were 1-1000 ng ml(-1) and 5-5000 ng g(-1) using 50 microl of mouse plasma and 50 microl of tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma), respectively. The method can be applied to studies generating only small sample volumes (e.g. mouse plasma and tumor tissue), but also to studies in human plasma requiring a lower limit of quantitation. The assay was applied successfully to several studies with both human and mouse samples.

Determination of free and total cyst(e)ine in plasma of dogs and cats

In human blood, the amino acid cysteine forms disulfide bonds with itself and with other sulfhydryl compounds in their free form and with sulfhydryls in protein. Protein-bound cysteine is lost when plasma proteins are removed before amino acid analysis. The purpose of this study was to assess the time course and extent of cyst(e)ine (cysteine + half-cystine) loss in dog and cat plasma. An equal volume of 6% sulfosalicylic acid was added to plasma aliquots at 0, 2, 4, 10, 16, 24, 36, 48, 60, and 72 hours after separation of blood cells. Tris-2-carboxyethyl-phosphine hydrochloride (TCEP - HCl), a reducing agent, was used to regenerate total plasma cyst(e)ine after 3 months of sample storage (-20 degrees C). Initial free cyst(e)ine concentrations (mean +/- SEM) were higher in canine plasma (77 +/- 4 micromol/L) than in feline plasma (37 +/- 3 micromol/L). Free plasma cyst(e)ine concentrations in dogs and cats decreased after first-order kinetics, with a half-life of 23 and 69 hours, respectively. Total plasma cysteine after TCEP - HCl treatment was similar for dogs (290 micromol/L) and cats (296 micromol/L), but the percentage of free cysteine was higher (P = .02) in dogs (27%) than in cats (13%). Over half of the cyst(e)ine, homocysteine, cysteinylglycine, and glutathione were bound in vivo to plasma proteins. These results emphasize the importance of removing plasma proteins within 1 hour after blood collection for reliable assay of free plasma cyst(e)ine.

Private sector hospital response to the 2003 dengue outbreak in the Indian capital metropolis of Delhi

During 2003, the Indian capital metropolis of Delhi was afflicted with an outbreak of dengue. A private-sector tertiary care hospital responded instantly to the sudden influx of 162 patients during an 8-week interval. That was an unusual challenge because, until 1997, the hospital had exclusively managed patients with ophthalmic disorders. Dengue patients received fiscal concessions and several free services. A multidisciplinary cell was commissioned for case management. Apart from a diagnosis of patients with dengue and their clinical management, suspected or confirmed dengue episodes were instantly reported to health authorities. Anti-mosquito measures in the hospital premises including residential areas for the nursing personnel prevented any local virus transmission. The integrated therapeutic and public health response was associated with a 1.23% case fatality rate. The protocol developed during the dengue outbreak would address every locally reportable disease in the future.

Assay of plasma semicarbazide-sensitive amine oxidase and determination of its endogenous substrate methylamine by liquid chromatography

Semicarbazide-sensitive amine oxidase (SSAO) is present in plasma, as well as in other tissues. Previous studies indicated that SSAO is of important physiological and pathophysiological functions. HPLC methods were developed for the assay of SSAO in plasma, and for the determination of plasma methylamine, an SSAO's endogenous substrate. Benzylamine was used as artificial substrate for the enzyme activity assay of SSAO. A 0.2-ml aliquot of plasma was incubated with benzylamine at 37 °C for 30 min. Benzaldehyde, the enzymatic reaction product, was derivatized with 2,4-dinitrophenylhydrazine (DNPH), and analyzed with HPLC and UV detection. SSAO enzyme activity is defined as benzaldehyde (nmol) formed per ml plasma per hour. Recoveries of benzaldehyde spiked to plasma were between 63.5 and 68.2% with relative standard deviation less than 3%. To determine methylamine in plasma, a 0.1-ml aliquot of plasma was deproteinized by trichloroacetic acid and centrifugation. The supernatant was derivatized with dansyl chloride and analyzed by HPLC with fluorescence detection. Recoveries of spiked methylamine at ppb (ng/ml) level were between 93.7 and 97.6% with relative standard deviation less than 2.5%.

Quantification of a novel retinoic acid metabolism inhibitor, 4-(1H-imidazol-1-yl)retinoic acid (VN/14-1RA) and other retinoids in rat plasma by liquid chromatography with diode-array detection

A simple reversed phase high performance liquid chromatographic (HPLC) method was developed for the separation and quantification of a novel retinoic acid metabolism inhibitor, 4-(1H-imidazol-1-yl)retinoic acid (VN/14-1RA), and other retinoids in rat plasma. VN/14-1RA, alone or in combination with ATRA, is effective at inhibiting the proliferation of prostate and breast cancer cell lines in vitro. Aliquots of rat plasma were spiked with the retinoids followed by addition of acetonitrile for precipitation of plasma proteins. The decanted supernatant was evaporated under a stream of nitrogen and reconstituted in acetonitrile. Analysis was accomplished by injection of an aliquot of the reconstituted sample into an HPLC system consisting of a Zorbax Rx-C18 column and a diode array detector. A mobile phase composed of ammonium acetate (0.1 M), acetic acid solution (2% (v/v)) and methanol at a flow rate of 1.0 mL/min was used for gradient elution. The recoveries for all compounds ranged from 65 to 85% regardless of the concentrations examined. The HPLC assay was linear over the range 0.10–5.0 μg/mL (CV < 10%) with a limit of quantification of 100 ng/mL for VN/14-1RA. A one-compartment model with apparent first-order elimination was used to describe the plasma concentration-time profile for VN/14-1RA after intravenous administration. The mean terminal elimination half-life (t1/2) was 19.0 ± 3.2 min. This HPLC method is useful for the analysis and evaluation of the pharmacokinetics of VN/14-1RA in rats.

Influence of Storage Time and Temperature on International Normalized Ratio (INR) Levels and Plasma Activities of Vitamin K Dependent Clotting Factors

We designed this study to assess the effect of storage time and temperature on the international normalized ratio (INR) levels and plasma activities of vitamin K-dependent, clotting factors. A total of 100 subjects, comprising 34 healthy controls, 33 patients with liver cirrhosis and 33 patients on long-term coumarin therapy were enrolled. After centrifugation of collected specimens, aliquots of plasma were stored at room temperature (20 −22°C), refrigerated at 2-6°C and frozen at −40°C. Determinations of INR and plasma activities of clotting factors II, VII, IX and X were performed immediately after sampling (0 time) and after 6, 12 and 24 h. We found no significant change of either INR levels or plasma activity of any of the studied clotting factors up-to 24 h at different studied temperatures ( p >0.05). Our data demonstrates that clinical specimens for determination of INR levels and plasma activities of factors II, VII, IX and X are acceptable for testing for up-to 24 h whatever may be the temperature of storage.

Quantification of a novel retinoic acid metabolism inhibitor, 4-(1H-imidazol-1-yl)retinoic acid (VN/14-1RA) and other retinoids in rat plasma by liquid chromatography with diode-array detection

A simple reversed phase high performance liquid chromatographic (HPLC) method was developed for the separation and quantification of a novel retinoic acid metabolism inhibitor, 4-(1H-imidazol-1-yl)retinoic acid (VN/14-1RA), and other retinoids in rat plasma. VN/14-1RA, alone or in combination with ATRA, is effective at inhibiting the proliferation of prostate and breast cancer cell lines in vitro. Aliquots of rat plasma were spiked with the retinoids followed by addition of acetonitrile for precipitation of plasma proteins. The decanted supernatant was evaporated under a stream of nitrogen and reconstituted in acetonitrile. Analysis was accomplished by injection of an aliquot of the reconstituted sample into an HPLC system consisting of a Zorbax Rx-C18 column and a diode array detector. A mobile phase composed of ammonium acetate (0.1 M), acetic acid solution (2% (v/v)) and methanol at a flow rate of 1.0 mL/min was used for gradient elution. The recoveries for all compounds ranged from 65 to 85% regardless of the concentrations examined. The HPLC assay was linear over the range 0.10–5.0 μg/mL (CV < 10%) with a limit of quantification of 100 ng/mL for VN/14-1RA. A one-compartment model with apparent first-order elimination was used to describe the plasma concentration-time profile for VN/14-1RA after intravenous administration. The mean terminal elimination half-life ( t 1/2) was 19.0 ± 3.2 min. This HPLC method is useful for the analysis and evaluation of the pharmacokinetics of VN/14-1RA in rats.

Assay of plasma semicarbazide-sensitive amine oxidase and determination of its endogenous substrate methylamine by liquid chromatography

Semicarbazide-sensitive amine oxidase (SSAO) is present in plasma, as well as in other tissues. Previous studies indicated that SSAO is of important physiological and pathophysiological functions. HPLC methods were developed for the assay of SSAO in plasma, and for the determination of plasma methylamine, an SSAO's endogenous substrate. Benzylamine was used as artificial substrate for the enzyme activity assay of SSAO. A 0.2-ml aliquot of plasma was incubated with benzylamine at 37 °C for 30 min. Benzaldehyde, the enzymatic reaction product, was derivatized with 2,4-dinitrophenylhydrazine (DNPH), and analyzed with HPLC and UV detection. SSAO enzyme activity is defined as benzaldehyde (nmol) formed per ml plasma per hour. Recoveries of benzaldehyde spiked to plasma were between 63.5 and 68.2% with relative standard deviation less than 3%. To determine methylamine in plasma, a 0.1-ml aliquot of plasma was deproteinized by trichloroacetic acid and centrifugation. The supernatant was derivatized with dansyl chloride and analyzed by HPLC with fluorescence detection. Recoveries of spiked methylamine at ppb (ng/ml) level were between 93.7 and 97.6% with relative standard deviation less than 2.5%.

Determination of fraction unbound docetaxel using microequilibrium dialysis

402 Docetaxel (Taxotere) has been shown to bind extensively in vitro to serum proteins, including α1-acid glycoprotein (AAG), albumin, and lipoproteins. High AAG levels have been associated with both a decrease in the unbound drug fraction in vitro and a decrease in total docetaxel clearance in vivo. It has also been recently reported that the vascular binding of docetaxel is influenced by its formulation vehicle polysorbate 80 in a concentration-dependent manner (Loos et al., Clin. Pharmacol. Ther. 74: 364-371, 2003). In an effort to better understand the role of polysorbate 80 and serum proteins in docetaxel pharmacology, we have developed and validated a novel, rapid and reproducible assay based on micro-equilibrium dialysis for measuring the fraction unbound (fu) docetaxel in patient plasma samples. Equilibrium dialysis was performed on a rotator at 37°C in a humidified atmosphere of 5% CO2 using 96-wells micro-dialysis plates. The dialysis compartments in each well were separated by a regenerated cellulose membrane with a 5 kDa cut-off. Experiments were carried out with 250-μl aliquots of plasma containing a tracer amount of [3H] docetaxel with high-specific activity against an equal volume of 0.01 M phosphate buffer (pH 7.4). Drug concentrations in both compartments were measured by liquid scintillation counting for 1 minute. Equilibrium for docetaxel transport across the membrane was established within 4 hours. Because the volume shift during dialysis was negligible (

Simultaneous selected reaction monitoring, MS/MS and MS3 quantitation for the analysis of pharmaceutical compounds in human plasma using chip‐based infusion

An assay method with mass spectrometric detection was developed for the quantitative analysis of a pharmaceutical compound and its major metabolite in human plasma using chip-based infusion. Liquid-liquid extraction sample preparation was found to be essential to minimize matrix suppression and to achieve a limit of quantitation (LOQ) of 2.5 ng/mL using a 100 microL plasma aliquot. The potential for simultaneous quantitation in selected reaction monitoring (SRM), tandem mass spectrometry (MS/MS) (enhanced product ion), and MS(3) was investigated and found to be very beneficial in improving assay selectivity. A novel concept for monitoring quantitative assay performance using a SRM/MS(3) ratio is proposed.

The reproducibility of human platelet-rich plasma with respect to platelet counts and putative cytokine levels

Statement of the Problem: Platelet-rich plasma (PRP) has been advocated as a beneficial adjunct to bone grafting procedures in the maxillofacial region. Many questions remain unanswered, however, concerning PRP efficacy when used in combination with bone grafting. This study sought to establish what range of PRP concentrates and cytokine levels could predictably be obtained from healthy volunteers using a commercially available automated centrifuge designed specifically for the collection of PRP. Materials and Methods: Twenty-four blood samples were collected from 15 male and 9 female healthy volunteers. All samples (60 mL for females, 55 mL for males) had a 500 microliter aliquot removed for determination of baseline complete blood counts. A SmartPReP machine (Harvest Technologies) was used according to the manufacturer’s instructions to prepare the PRP. Aliquots of platelet-poor plasma (PPP), PRP, and whole blood were run on the Coulter STKS (Coulter Corporation). Eleven PRP samples underwent cytokine analysis. Levels of platelet derived growth factor AB (PDGF-AB) were determined using a Human PDGF-AB Quantikine Colorimetric Sandwich ELISA kit (R&D Systems). Transforming growth factor beta 1 (TGF-β1) levels were determined using a Human TGF-β1 Colorimetric Sandwich ELISA kit (YesBiotech). Method of Data Analysis: All quantitative measurements were described using summary statistics. Scatterplots and Spearman rank correlation coefficient were used to identify relationships between 1) platelet counts in whole blood and PRP, and 2) growth factor levels and platelet counts. Student’s t tests were also performed to identify any differences in test groups. Results: Analysis of PRP samples showed an average platelet increase of 396%. Significant variation did exist, however, with platelet counts ranging from 363 × 109 cells/L to 1,900 × 109 cells/L. Females did appear to produce a more concentrated PRP product, but this was not statistically significant. An association was demonstrated between the volunteer’s whole blood platelet count and that of the PRP. A high degree of variability was also noted when examining the results of the initial cytokine release levels of PRP and PPP. Although variability did exist, relative cytokine increases in PRP compared to PPP were 151% for TGF-β1 and 415% for PDGF-AB. No correlation was noted between growth factor levels and platelet counts. Conclusion: These initial results demonstrate increases in both platelet counts and cytokine levels. Of concern, however, was the significant variation in these levels among PRP samples obtained from healthy volunteers using a commercially available device. References Marx RE, Carlson ER, Eichstaedt RM, et al: Platelet rich plasma: Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:638, 1998 Weibrich G, Kleis WK, Hafner G: Growth factor levels in the platelet rich plasma produced by 2 different methods: Curasan-type PRP kit versus PCCS PRP system. Int J Oral Maxillofac Implants 17:184, 2002

Plasma kinetics and uptake of a cholesterol-rich microemulsion (LDE) associated to etoposide oleate by neoplastic ovarian tissues

3176 Background: Previously we showed that etoposide oleate (ETO)associated with a cholesterol-rich microemulsion (LDE) forms a stable complex that is taken up by malignant cells. LDE-ETO retains the in vitro anti-proliferative activity of the drug and markedly reduces the toxicity to animals. In this study, the plasma kinetics of LDE:ETO and the ability of the complex to concentrate in the tumor after injection into the patients were tested. Methods: LDE-ETO labeled with [3H]etoposide oleate and [14C]-Cholesteryl Oleate (CO) was intravenously injected into 10 patients with ovarian cancer (50,6 ± 7,5 yr.) 24 h before the surgery. Blood samples were collected over the 24 h period to determine the plasma decay curves of the complex labels. Radioactivity present in plasma aliquots was determined in a scintillation solution and the plasma fractional clearance rate (FCR) was calculated by compartmental analysis. Specimens of tumors and normal ovaries excised during the surgery were collected for lipid extraction, separation by thin layer chromatography and radioactive counting. Results: FCR of LDE and of the drug were similar (0,0881 e 0,1722, respectively, p = 0.0653). The mean uptake of both [14C]-LDE and [3H]-etoposide oleate by malignat tissue of ovary was three fold greater when compared with that of the normal contralateral normal ovaries (LDE uptake = 448 ± 184 and 143 ± 51 and etoposide oleate uptake = 346 ± 75 and 103 ± 56, respectively). Conclusions: Our results indicate that most of the drug remains associated with LDE particles while in the circulation and that LDE has the hability to target etoposide oleate to the malignant ovarian tissues. Thus, LDE-ETO is an interesting preparation for testing in clinical trials. No significant financial relationships to disclose.

Plasma kinetics and uptake of a cholesterol-rich microemulsion (LDE) associated to etoposide oleate by neoplastic ovarian tissues

3176 Background: Previously we showed that etoposide oleate (ETO)associated with a cholesterol-rich microemulsion (LDE) forms a stable complex that is taken up by malignant cells. LDE-ETO retains the in vitro anti-proliferative activity of the drug and markedly reduces the toxicity to animals. In this study, the plasma kinetics of LDE:ETO and the ability of the complex to concentrate in the tumor after injection into the patients were tested. Methods: LDE-ETO labeled with [3H]etoposide oleate and [14C]-Cholesteryl Oleate (CO) was intravenously injected into 10 patients with ovarian cancer (50,6 ± 7,5 yr.) 24 h before the surgery. Blood samples were collected over the 24 h period to determine the plasma decay curves of the complex labels. Radioactivity present in plasma aliquots was determined in a scintillation solution and the plasma fractional clearance rate (FCR) was calculated by compartmental analysis. Specimens of tumors and normal ovaries excised during the surgery were collected for lipid extraction, separation by thin layer chromatography and radioactive counting. Results: FCR of LDE and of the drug were similar (0,0881 e 0,1722, respectively, p = 0.0653). The mean uptake of both [14C]-LDE and [3H]-etoposide oleate by malignat tissue of ovary was three fold greater when compared with that of the normal contralateral normal ovaries (LDE uptake = 448 ± 184 and 143 ± 51 and etoposide oleate uptake = 346 ± 75 and 103 ± 56, respectively). Conclusions: Our results indicate that most of the drug remains associated with LDE particles while in the circulation and that LDE has the hability to target etoposide oleate to the malignant ovarian tissues. Thus, LDE-ETO is an interesting preparation for testing in clinical trials. No significant financial relationships to disclose.

Determination of NG, NG-dimethyl- l-arginine, an endogenous NO synthase inhibitor, by gas chromatography–mass spectrometry

A fully validated gas chromatographic–mass spectrometric (GC–MS) method for the accurate and precise quantification of N G, N G-dimethyl- l-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of the NO synthase, in cell culture supernatants and in small volumes of plasma is described. ADMA was concentrated by solid phase extraction and converted to its methyl ester pentafluoropropionic amide derivative. The derivatives were analyzed without any further purification. Using gas chromatography–chemical ionization mass spectrometry, fragment ions at m/ z 634 and m/ z 640 were obtained for ADMA and for N G, N G-[ 2H 6]-dimethyl- l-arginine ([ 2H 6]-ADMA) as internal standard, respectively. [ 2H 6]-ADMA was synthesized by reaction of l-ornithine fastened at bromcyan-agarose with dimethylamine. The limit of detection of the method was 2 fmol, while the limit of quantitation for cell culture supernatants was 0.05 μM. The method was validated in a concentration range of 0–1.2 μM in cell culture medium and 0–2 μM in 50 μl aliquots of human plasma. The precision was ≥97% and the accuracy was determined to be ≥94%. This method is fast, rugged and an alternative to high performance liquid chromatography (HPLC) analysis of ADMA in cell culture supernatants and small volumes of human plasma.