Development of a microfluidic-chip system for liquid-phase microextraction based on two immiscible organic solvents for the extraction and preconcentration of some hormonal drugs.

In the present study, for the first time, an on-chip liquid phase microextraction (LPME) coupled with high performance liquid chromatography was introduced for the analysis of levonorgestrel (Levo), dydrogesterone (Dydo) and medroxyprogesterone (Medo) as the model analytes in biological samples. The chip-based LPME set-up was composed of two polymethyl methacrylate (PMMA) plates with microfabricated channels and a microporous membrane sandwiched between them to separate the sample solution and acceptor phase. These channels were used as a flow path for the sample solution and a thin compartment for the acceptor phase, respectively. In this system, two immiscible organic solvents were used as supported liquid membrane (SLM) and acceptor phase, respectively. During extraction, the model analytes in the sample solution were transported through the SLM (n-dodecane) into the acceptor organic solvent (methanol). The new set-up provided effective and reproducible extractions using low volumes of the sample solution. The effective parameters on the extraction efficiency of the model analytes were optimized using one variable at a time method. Under the optimized conditions, the new set-up provided good linearity in the range of 5.0-500µgL(-1) for the model analytes with the coefficients of determination (r(2)) higher than 0.9909. The relative standard deviations (RSDs%) and limits of detection (LODs) values were less than 6.5% (n=5) and 5.0µgL(-1), respectively. The preconcentration factors (PFs) were obtained using 1.0mL of the sample solution and 20.0µL of the acceptor solution higher than 19.9-fold. Finally, the proposed method was successfully applied for the extraction and determination of the model analytes in urine samples.

Evaluation of storage and evaporation in the removal efficiency of D-norgestrel and progesterone in human urine.

Pharmaceuticals are emerging contaminants and it must be noted that approximately 70 % of them are excreted via urine. Therefore, urine usage implies the risk of transfer of pharmaceutical residues to agricultural fields and environment contamination. Thus, this study aimed on the development and validation of a LC-MS/MS method for D-norgestrel (D-NOR) and progesterone (PRO) determination in human urine, as well as the evaluation of the removal efficiency of two methods (storage and evaporation), and the effects of acidification with sulfuric acid. The storage process was evaluated for 6 weeks, while the evaporation was assessed at three different temperatures (50, 75, and 100 °C). All experiments were done with normal urine (pH = 6.0) and acidified urine (pH = 2.0, with sulfuric acid). The results of validation showed good method efficiency. In the second week of storage, higher hormone degradation was observed. In the evaporation method, both D-NOR and PRO were almost completely degraded when the volume was reduced to the lowermost level. Results also indicate that acidification did not affect degradation. Overall, the results showed that combination of two methods can be employed for more efficient hormone removal in urine.

Automated hollow fiber microextraction based on two immiscible organic solvents for the extraction of two hormonal drugs.

In this research, a rapid efficient and automated instrument based on hollow fiber liquid-phase microextraction (HF-LPME) followed by high performance liquid chromatography (HPLC) with UV-vis detection was applied for the preconcentration and determination of two hormonal drugs (megestrol acetate and levonorgestrel) in water and urinary samples. n-Dodecane was used as the supported liquid membrane (SLM) and methanol was used as the acceptor phase in the hollow fiber lumen. The effects of different parameters such as fiber length, extraction time, stirring rate, and ionic strength on the extraction efficiency were investigated using modified simplex and central composite design as the screening and optimization methods, respectively. The composition effect of SLM and type of acceptor phase were optimized separately. For adjustment of the SLM composition, trioctylphosphine oxide (TOPO) was chosen. Under optimized condition, the calibration curves were linear (r(2)>0.997) in the range of 0.5-200 µg L(-1). LOD for both of the drugs were 0.25 µg L(-1). The applicability of this technique was examined by analyzing drugs in water and urine samples. The relative recoveries of the drugs were in the range of 86.2-102.3% that show the capability of the method for the determination of the drugs in various matrices.

Effect of two oral contraceptives with different ethinyl estradiol and levonorgestrel concentrations on the urinary excretion of biochemical vasoactive markers.

In the present study the effect on the urinary excretion of vasoactive markers of two oral contraceptives (OCs), i.e., Leios, containing 0.02 mg ethinyl estradiol and 0.1 mg levonorgestrel, and Stediril 30, containing 0.03 mg ethinyl estradiol and 0.15 mg levonorgestrel, was investigated. cGMP, prostacyclin and its antagonist thromboxane, serotonin, and urodilatin, a natriuretic and diuretic peptide formed in the kidney, were measured as markers. In a comparative, double-blind, randomized, parallel group study, 34 women received Leios and 33 women Stediril 30. Nocturnal urine was collected before treatment and during cyclic treatment after 3 and 12 cycles. Both contraceptives significantly enhanced cGMP excretion after 12 cycles. The prostacyclin metabolite remained unchanged for both formulations, but the excretion of the thromboxane metabolite was significantly decreased after 12 cycles. Thus, the ratio of prostacyclin to thromboxane, crucial for the resulting effect on vascular tone, increased significantly. For the serotonin metabolite, no changes were observed for both contraceptives. The excretion of urodilatin significantly increased for both preparations after 12 cycles compared to the pretreatment values. These results indicate that the low-dose OCs Leios and Stediril 30 may stimulate the production of some vasoactive markers, at least after 12 cycles of treatment. The positive influence of these contraceptives on the various markers investigated may improve vascular tone, impede development of atherosclerosis and arterial thrombosis, and improve water and electrolyte homeostasis. These effects most likely can be attributed to the estrogenic component. Levonorgestrel may elicit no impact on these estrogen-induced changes that, however, seem only to be manifested after a longer treatment period.

An enzyme immunoassay for serum and urinary levonorgestrel in human and non-human primates.

A microtiter plate enzyme immunoassay (EIA) is reported for the measurement of levonorgestrel (LNG) in serum and urine samples of human and non-human primates, and the results are compared to data obtained by radioimmunoassay (RIA). Rabbit polyclonal antibodies were raised against the bovine serum albumin conjugate of the 3-O-carboxymethyl oxime (CMO) derivative of LNG. The enzyme label was produced by the conjugation of horseradish peroxidase to LNG at the 3-position by the same CMO bridge used for the immunogen. The assay requires 2.5 hours to perform using 2.2-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt as the chromogenic substrate. Serum (100 microliters) is extracted with petroleum ether prior to assay, whereas urine samples (25 microliters) are diluted and measured directly. The sensitivity of the assay is 0.25 pg/well with a 50% displacement of label at 7.5-9.5 pg and a linear response through 250 pg/well. Minimum levels of 8.7 and 10.0 pg/ml can be detected in serum and urine samples, respectively. Changes in serum LNG concentrations were measured in women and non-human primates following LNG implantation or injection. In the non-human primate study, serum LNG concentrations began to rise rapidly following i.m. injection of LNG, with peak levels occurring on days 3 to 5, then decreasing to approximately 25-35% of peak levels for the duration of the study. Circulating concentrations of 1.86 +/- 0.18 ng/ml LNG were reached in women the first week post-insertion of Norplant implants and decreased by 50% at 7-10 days, 75% after 14-21 days, followed by a steady decrease during the next 60-70 days to constant low levels that exhibited a high individual variation. Correlation coefficients of EIA and RIA results were 0.988 for human serum, 0.926 for human urine, and 0.972 for non-human primate serum.