An optimised, highly sensitive radioimmunoassay for the simultaneous measurement of estrone, estradiol and estrone sulfate in the ultra-low range in human plasma samples

Following the introduction of potent aromatase inhibitors for the treatment of breast cancer patients, highly sensitive methods have become mandatory to evaluate the influence of these drugs on plasma estrogen levels. Commercially available kits for estrogen measurements are not suitable for these kinds of evaluations due to their detection limits that are close to baseline estrogen levels in postmenopausal women. We describe here an optimised radioimmunoassay suitable for the simultaneous measurement of plasma estrone (E₁), estradiol (E₂) and estrone sulfate (E₁S) levels in the ultra-low range. Following incubation with [³H]-labelled estrogens as internal standards, crude estrogen fractions were separated by ether extraction. The E₁S fraction was hydrolysed with sulfatase followed by eluation on a Sephadex column. Free estrogens (E₁, E₂) were separated by chromatography (LH-20). Estrone and E₁S (following hydrolysis) were converted into E₂, and each estrogen fraction was measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2-[¹²⁵I]-iodo-histamine) as ligand. Although several purification steps were involved, the internal recovery values for tritiated estrogens were found to be 88%, 90%, and 49% for E₁, E₂ and E₁S, respectively. The intra-assay coefficient of variation was <5% for all recovery measurements. The detection limits were calculated following repeated blank measurements and found to be 1.14 pmol/L for E₁, 0.67 pmol/L for E₂, and 0.55 pmol/L for E₁S, respectively. The intra-assay coefficient of variation (CV) was found to be 3.4% for E₁, 5.1% for E₂ and 6.1% for E₁S, while the inter-assay CV was 13.6%, 7.6% and 7.5% for E₁, E₂, and E₁S, respectively. Considering normal plasma levels for E₂ (15 pmol/L), E₁ (80 pmol/L) and E₁S (400 pmol/L) in postmenopausal women, the method allows theoretically to detect suppression of plasma E₂, E₁ and E₁S levels by 95.5%, 98.6% and 99.9% when starting from average, normal postmenopausal levels. Thus, the method presented here is to our knowledge the currently most sensitive assay available for plasma estrogen measurements in the ultra-low range and, as such, a reliable tool for a proper evaluation of potent aromatase inhibitors and other potential drugs influencing on plasma estrogen levels.     

Determination of norgestimate and its metabolites in human serum using high-performance liquid chromatography with tandem mass spectrometric detection

A rapid and reliable analytical method is described for the simultaneous determination of a synthetic progestin norgestimate (NGM), and its metabolites, 17-deacetylnorgestimate (17-DA-NGM), 3-ketonorgestimate (3-keto-NGM) and norgestrel (NGL) in human serum using reversed phase high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. The assay was linear over the concentration ranges of 0.1–5.0 ng/ml for 17-DA-NGM and NGL and 0.5–5.0 ng/ml for NGM and 3-keto-NGM. The inter-assay reproducibility was consistently less than 10%. The overall recovery of the analytes ranged from 72 to 92%. Serum profiles following oral administration of norgestimate to female volunteers are presented.     

Determination of felodipine, its enantiomers, and a pyridine metabolite in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of racemic felodipine, its enantiomers, and a pyridine metabolite in human plasma are described. Following liquid-liquid extraction from plasma, enantiomers of felodipine were separated on a chiral HPLC column (Chiralcel OJ) and fractions containing each isomer were collected on a continuous basis using a fraction collector. These fractions were later analyzed by GC-MS-SIM. A similar method based on GC-MS-SIM detection was developed for the determination of racemic felodipine and its pyridine metabolite with a minor modification of sample preparation. The limits of quantitation in plasma were 0.1 ng/ml for both the R(+)- and S(−)-enantiomers of felodipine and 0.5 ng/ml for both racemic felodipine and its pyridine metabolite. The stereoselective assay was used to support a clinical study with racemic felodipine, and was capable of analyzing more than 30 plasma samples per day.     

Diaryl dimers of estradiol and of estrone may be formed as major metabolites by mouse mammary glands

The formation of a novel estrogen metabolite by mammary tissues was investigated. Polar and nonpolar metabolites of endogenous estrogens are formed in liver and other tissues. Polar products such as the catechol estrogens are implicated in tumorigenesis in breast tissue, whereas a nonpolar metabolite, 2-methoxyestradiol, may be protective. Diaryl ether dimers, as a novel form, have been reported as nonpolar products from liver microsomes. We have noted major amounts of nonpolar metabolites in other tissues that were neither 2-methoxyestrogens nor estrogen fatty acid esters. The possible formation of such novel metabolites by breast tissues from adult nulliparous mice with [³H]-labeled estrogens as substrates was considered. Steroids were recovered from media by solid-phase extraction and profiles were obtained from HPLC (acetonitrile:water). Saponification was done with an internal standard of estradiol stearate. Major amounts of nonpolar metabolites were formed in all instances, with one or two principal peaks. Alkaline hydrolysis had no effect on the nonpolar product(s) but released estradiol from its stearate. Strong acid treatment also had no effect as shown by HPLC. Thus, it is suggested that diaryl dimers of estrogens may be formed as major metabolites by mouse mammary glands.     

Determination of a cyclooxygenase II inhibitor in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of a cyclooxygenase II (COX-II) inhibitor (3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5'-dimethyl-5H-furan-2-one, I) in human plasma, in two concentration ranges of 0.1-20 and 5-1000 ng/ml, are described. Following liquid-liquid extraction, the residue, after evaporation of the organic phase to dryness, was reconstituted in acetonitrile (20 l) and part of the extract (1 l) was analyzed by GC/MS/SIM. The drug (I) and internal standard (II) were separated on a 25 mx0.2 mm capillary column with HP Ultra 1 (100% dimethylpolysiloxane, 0.33 m) phase and analyzed by MS/SIM monitoring ions at m/z 237 and 282 for I and II, respectively. The standard curve was linear within the lower concentration range of 0.1-20 ng/ml and the lower limit of quantification (LLOQ) in plasma was 0.1 ng/ml. Intraday coefficients of variation (CV, n=5) were 8.9, 4.2, 5.7, 3.1, 1.9, 1.9, and 4.4% at 0.1, 0.2, 0.5, 1.0, 5.0, 10, and 20 ng/ml, respectively. The standard curve was also linear within the higher concentration range of 5-1000 ng/ml and the LLOQ in plasma was 5 ng/ml. Intraday coefficients of variation (CV, n=5) were all below 9% at all concentrations within the standard curve range. The accuracy for I in human plasma was 91-112% and the recovery of I and II was greater than 70% at all concentrations within both standard curve ranges. The details of the assay methodology are presented.     

Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

Determination of dihydroergotamine in human plasma by high-performance liquid chromatography with fluorescence detection

Dihydroergotamine, a 5-hydroxytryptamine antagonist, is used for the treatment of vascular headaches. A high-performance liquid chromatography assay with fluorescence detection is described for the determination of dihydroergotamine in plasma. The assay was validated over the concentration range 0.1–10 ng/ml plasma and applied to the analysis of plasma samples from subjects treated intramuscularly and intranasally with 2 mg of dihydroergotamine.     

Determination of the antidepressant agent citalopram and metabolites in plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the determination of citalopram [1-(3-(dimethylaminopropyl)-1-(4-fluorophenyl)-5-phthalancarbonitrile] and its two main metabolites (the methylamino and amino derivatives). The compounds were extracted from alkaline plasma with diethyl ether. The combined ether layers were evaporated after addition of 50 μl of 0.1 N HCl. The residual extracts were purified with diethyl ether and 20 μl were injected into a Spherisorb ODS 5-μm column with acetonitrile–0.6% phosphate buffer pH 3 (55:45, v/v) as the mobile phase. Using a fluorescence detector the detection limits are 1 ng/ml of plasma for citalopram and the methylamino metabolite and 0.5 ng/ml for the amino metabolite.     

Shp2 protein tyrosine phosphatase inhibitor activity of estramustine phosphate and its triterpenoid analogs

Shp2 protein tyrosine phosphate (PTP) is a novel target for anticancer drug discovery. We identified estramustine phosphate as a Shp2 PTP inhibitor from the National Cancer Institute Approved Oncology Drug set. A focused structure-activity relationship study indicated that the 17-phosphate group is required for the Shp2 PTP inhibitor activity of estramustine phosphate. A search for estramustine phosphate analogs led to identification of two triterpenoids, enoxolone, and celastrol, having Shp2 PTP inhibitor activity. With the previously reported PTP1B inhibitor trodusquemine, our study reveals steroids and triterpenoids with negatively charged phosphate, carboxylate, or sulfonate groups as novel pharmacophores of selective PTP inhibitors.     

Determination of amidepin in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection

An isocratic reversed-phase high-performance liquid chromatographic method for the determination of amidepin has been developed. The method is based on the extraction of alkaline plasma with diethyl ether—dichloromethane, and the injection into the Supelcosil LC-18 column of the evaporated and reconstituted organic phase. After separation, detection is carried out by a fluorescence detector (excitation at 195 nm with no filter). The limit of detection is 10 ng/ml of plasma. The mean coefficient of variation is 12%. The plasma levels after oral administration and after intravenous administration are shown.     

Determination of terazosin in human plasma, using high-performance liquid chromatography with fluorescence detection

A selective, sensitive, rapid and reproducible high-performance liquid chromatographic method for the determination of terazosin in plasma is described. The structurally related compound prazosin was used as an internal standard. The method comprises extraction with methylene chloride followed by chromatography on a C₁₈ reversed-phase column. The compounds were detected using spectrofluorimetry. The absolute recoveries were more than 90% with a minimal detection of 1 ng/ml and calibration curve was linear between 1 and 80 ng/ml.     

Determination of vigabatrin in human plasma and urine by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of vigabatrin in human plasma and urine. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan, solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Aspartam was used as an internal standard. The assay was linear over the concentration range of 0.2–20.0 μg/ml for plasma and 1.0–15.0 μg/ml for urine with a lower limit of detection of 0.1 μg/ml using 0.1 ml of starting volume of the sample. Both the within-day and day-to-day reproducibilities and accuracies were less than 5.46% and 1.6%, respectively. After a single oral dose of 500 mg of vigabatrin, the plasma concentration and the cumulative urinary excretion of the drug were determined.     

Simultaneous determination of hydrocodone and hydromorphone in human plasma by liquid chromatography with tandem mass spectrometric detection

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the simultaneous analysis of hydrocodone (HYC) and its metabolite hydromorphone (HYM) in human plasma. A robotic liquid handler and a 96-channel liquid handling workstation were used to aliquot samples, to add internal standard (I.S.), and to extract analytes of interest. A 96-well mixed-mode solid-phase cartridge plate was used to extract the analytes and I.S. The chromatographic separation was on a silica column (50 x 3 mm, 5-microm) with a mobile phase consisting of acetonitrile, water and trifluoroacetic acid (TFA) (92:8:0.01, v/v). The run time for each injection was 2.5 min with the retention times of approximately 2.1 and 2.2 min for HYC and HYM, respectively. The tandem mass spectrometric detection was by monitoring singly charged precursor-->product ion transition 300-->199 (m/z) for HYC, and 28-->185 (m/z) for HYM. The validated calibration curve range was 0.100-100 ng/ml, based on a plasma volume of 0.3 ml. The correlation coefficients were greater than or equal to 0.9996 for both HYC and HYM. The low limit of quantitation (LLOQ) was 0.100 ng/ml for both HYC and HYM with signal-to-noise ratio (S/N) of 50 and 10. respectively. The deuterated analytes, used as internal standards, were monitored at mass transitions 303-->199 (m/z) for HYC-d3 and 289-->185 (m/z) for HYM-d3. The inter-day (n= 17) precision of the quality control (QC) samples were < or = 3.5% RSD (relative standard deviation) for HYC and < or = 4.7% RSD for HYM, respectively. The inter-day accuracy of the QC samples were < or = 2.1% RE (relative error) for HYC and < or = 1.8% RE for HYM. The intra-day (n=6) precision and accuracy of the QC samples were < or = 2.6% RSD and < or = 3.0% RE for HYC, and < or = 4.7% RSD and < or = 2.4% RE for HYM. There was no significant deviation from the nominal values after a 5-fold dilution of high concentration QC samples by blank matrix. The QC samples were stable when kept at room temperature for 24-h or experienced three freeze-thaw cycles. The extraction recoveries were 86% for HYC and 78% for HYM. No detectable carryover was observed when a blank sample was injected immediately after a 2500 ng/ml sample that was 25-fold more concentrated than the upper limit of quantitation (ULOQ).     

Quantification of ketotifen and its metabolites in human plasma by gas chromatography mass spectrometry

Gas chromatography mass spectrometry with selected ion monitoring has been used to develop a method for the quantification of ketotifen and its demethylated, 10-hydroxy and 10-hydroxy demethylated metabolites in human plasma. The minimum detectable concentrations for ketotifen and its demethylated metabolites were 50 pg ml-1 and 300 pg ml-1 for the 10-hydroxy metabolite. The methodology has been applied in studies of the kinetics of the drug in man, and plasma levels of the unchanged drugs and its metabolites in free and conjugated form are reported.     

Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection

Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4 pmol of homocysteine and is highly reproducible (intra- and interassay coefficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.     

Determination of tianeptine in human plasma using high-performance liquid chromatography with fluorescence detection

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C(18) column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-10mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5-300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5mg TIA in a healthy volunteer.     

Determination of montelukast sodium in human plasma by column-switching high-performance liquid chromatography with fluorescence detection

MK-0476 (montelukast sodium) is a potent and selective cysteinyl leukotriene receptor antagonist that is being investigated in the treatment of asthma. A simple and sensitive method for the determination of MK-0476 in human plasma was developed using column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. A plasma sample was injected directly onto the HPLC system consisting of a pre-column (Capcell pak MF) and an analytical column (Capcell pak C18) which were connected with a six-port switching valve. The column eluate was monitored with a fluorescence detector (excitation at 350 nm; emission at 400 nm). The calibration curve was linear in a concentration range of 1–500 ng ml⁻¹ for MK-0476 in human plasma. The intra-day coefficients of variation of all concentrations within the range was less than 9.2%, and the intra-day accuracy values were between 97.2 and 114.6%. This method was used to measure the plasma concentration of MK-0476 following oral administration of the drug in humans.     

Determination of estramustine and its 17-keto metabolite in plasma by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of estramustine and its 17-keto metabolite in plasma. The assay involves extraction of the compounds into hexane from plasma buffered to pH 9.0, the residue obtained by evaporation of the hexane extract is dissolved in the mobile phase hexane—ethanol (92.5:7.5) with HPLC analysis performed on a 5-μm silica gel column using a fluorescence detector with excitation at 195 nm and emission at wavelengths greater than 250 nm. The overall recoveries and limits of sensitivity for estramustine and the 17-keto metabolite are 74.7% and 40 ng/ml of plasma and 85.1% and 50 ng/ml of plasma, respectively. The method was used to obtain plasma concentration—time profiles in three subjects with prostatic cancer following oral administration of a single 7 mg/kg dose of estramustine phosphate.     

Determination of esomeprazole and its two main metabolites in human, rat and dog plasma by liquid chromatography with tandem mass spectrometry

A LC-MS/MS method was developed for quantitative determination of esomeprazole, and its two main metabolites 5-hydroxyesomeprazole and omeprazole sulphone in 25 microL human, rat or dog plasma. The analytes and their internal standards were extracted from plasma into methyl tert-butyl ether - dichloromethane (3:2, v/v). After evaporation and reconstitution of the organic extract the analytes were separated on a reversed-phase LC column and measured by atmospheric-pressure positive ionisation MS. The linearity range was 20-20,000 nmol/L for esomeprazole and omeprazole sulphone, and 20-4000 nmol/L for 5-hydroxyesomeprazole. The extraction recoveries ranged between 80 and 105%. The intra- and inter-day imprecision were less than 9.5% with accuracy between 97.7% and 100.1% for all analytes.