Evaluation of a Hollow-Fiber Liquid-Phase Microextraction Technique for the Simultaneous Determination of PPI Drugs in Human Plasma by LC-DAD

Evaluation of a Hollow-Fiber Liquid-Phase Microextraction Technique for the Simultaneous... Abstract This study involved the development, validation and application of a three-phase hollow-fiber liquid-phase microextraction (HF-LPME) and liquid chromatography with diode array detection (LC-DAD) method for the simultaneous determination of the proton pump inhibitor (PPI) drugs omeprazole, pantoprazole and lansoprazole in human plasma. The evaluation of the HF-LPME parameters was crucial for the determination of the drugs and the conditions selected were: 1-octanol as solvent; phosphate buffer at pH 5 as donor phase; borate buffer at pH 10 as acceptor phase; extraction time of 15 min; stirring at 750 rpm and NaCl was added at 5% (w/v). Validation of the method according to US-FDA recommendations showed a good linear range (0.2–2.0 μg/mL) for all analytes, with a determination coefficient >0.9910. Precision was evaluated using intra- and inter-day assays, which showed relative standard deviations (RSD), <15% for all concentrations, with a limit of quantification (LOQ) of 0.2 μg/mL. Accuracy was also assessed at these concentration levels and was in the range from 80 to 130%. Finally, the sensitive, selective and reproducible HF-LPME/LC-DAD developed method was successfully applied to human plasma samples from patients undergoing therapy with the PPI drugs. Introduction Proton pump inhibitors (PPI) are some of the most widely prescribed drugs used in the treatment of disorders related to gastric acid secretion (1). PPIs, which include omeprazole (OME), pantoprazole (PAN) and lansoprazole (LANSO), are weak basic prodrugs that are converted to their active form in acid conditions. These activated forms block the gastric H+/K+ATPase (proton pump enzyme) which is responsible for the final step of the acid secretion process (2–4). As they have pKa1 and pKa2 values around 4 and 9, respectively (1), their activation can occur in the acid conditions of the stomach parietal cells (pH 1.3) that is their site of action (5). This is reflected in the fact that this class of drugs is more effective than others in the suppression of acid secretion (6). OME, PAN and LANSO have the same general mechanism of action and are unstable in acid environments but their different substituents on the pyridine and benzimidazole groups (Figure 1) result in some distinct properties (7). Concerning their acid stability, for example, the degradation rate is different and the stability order is: PAN > OME > LANSO (3). Another pharmacokinetic difference is the time taken to reach peak plasma concentration that is in the range of 0.5–3.5 h for OME, 1.7 h for LANSO and 1.1–3.1 h for PAN (8). All PPIs are rapidly absorbed and are highly plasma protein bound, which results in a low volume of distribution (9). The proportion of the drug bound to protein is 95% for OME, 98% for LANSO and 97% for PAN (8, 10, 11). Figure 1. View largeDownload slide Chemical structure of selected PPI drugs. Figure 1. View largeDownload slide Chemical structure of selected PPI drugs. Recent studies have made an association between chronic PPI therapy and potential adverse effects such as dementia, an increased risk of bone fractures and deficiency of vitamin B12 among others (12–15). For this reason, it is important to develop new methodologies to determine these drugs in biological matrices in order to evaluate their effects. In the literature, there are few works where the simultaneous determination of PPI drugs in human plasma has been performed, as reported by Bharathi and coworkers (2). Most studies concern one PPI drug and its enantiomers with their respective metabolites. They are usually determined in human plasma using liquid–liquid extraction (LLE) (2, 16, 17) or solid-phase extraction (SPE) (18) with liquid chromatography, usually coupled with tandem mass spectrometry (LC-MS/MS) or ultraviolet detection (LC-UV or LC-DAD). However, these conventional extraction methods have some disadvantages, such as the consumption of large volumes of toxic organic solvents, the requirement of costly cartridges and they are time-consuming (19–21). The hollow-fiber liquid-phase microextraction (HF-LPME) technique has received some attention since it uses low volumes of organic solvents to extract organic compounds from small amounts of aqueous samples (22). Furthermore, the technique employs a porous hydrophobic hollow fiber. This inexpensive disposable device eliminates the carry-over effect and its pore size can block the passage of larger molecules such as plasma proteins (23, 24). HF-LPME was introduced in 1999 (24) and performs the extraction of compounds from a donor phase (sample) through an organic solvent (extractor phase) into an acceptor phase inside the lumen of a hollow fiber. This acceptor phase can be the same extractor organic solvent as that immobilized in the fiber pores, corresponding to the two-phase mode; alternatively, it can be an aqueous solution when the technique is used in the three-phase mode (20). Because the extractor and acceptor phases are, respectively, immobilized in the pores and inside the lumen, the sample can be stirred or vibrated to accelerate the reaction kinetics without the loss of the phases (25, 26). There have been several reports of HF-LPME applications for drug and metabolite extraction from biological matrices (19, 27, 28). Concerning PPI drugs, to our knowledge, there is no documented HF-LPME method for the simultaneous determination of OME, PAN and LANSO in human plasma samples. Differently to previous studies concerning the determination of PPI drugs, in this paper we developed a miniaturized method (HF-LPME) for the simultaneous determination of three PPI drugs in human plasma by LC-DAD: OME, PAN and LANSO. The chromatographic and extraction parameters were evaluated in order to enable the extraction of these acid labeled and highly protein bound drugs from this complex matrix. Moreover, the method was validated and applied to human plasma samples from patients undergoing PPI therapy. Experimental Reagents OME, PAN, LANSO and sulfamethoxazole (internal standard, IS) analytical standards were purchased from Fluka Sigma-Aldrich® (São Paulo, Brazil). Monobasic potassium phosphate and high performance liquid chromatography (HPLC) grade acetonitrile and methanol were obtained from J.T. Baker® (Phillipsburg, USA). Dibasic potassium phosphate (Vetec®, Rio de Janeiro, Brazil) and boric acid (Isofar®, Rio de Janeiro, Brazil) were used to prepare buffer solutions in ultrapure water purified by a Milli-Q Millipore® system (18 MΩ at 25°C) (São Paulo, Brazil). Working standard drug solutions were prepared by the diluting 1 mg/mL methanol stock solutions to the appropriate volume with methanol and kept at −18°C protected from light. Hexane, toluene and 1-octanol were purchased from Sigma-Aldrich® (São Paulo, Brazil). Butyl acetate (F. Maia®, São Paulo, Brazil) and dichloromethane (Vetec®, Rio de Janeiro, Brazil) were also evaluated as organic solvents. pH adjustments were made using diluted solutions of sodium hydroxide (Proquímios®, Rio de Janeiro, Brazil) and hydrochloric acid (Dinâmica®, São Paulo, Brazil). Reagents used for synthetic plasma were all of analytical grade. Instrumentation and chromatographic conditions Chromatographic analyses were performed using an Agilent® 1260 Infinity HPLC system with quaternary pump and diode array detection. The separation was achieved using an Agilent® Microsorb MV100-5 C18 column (250 mm × 4.6 mm, 5 μm), preceded by an Agilent® guard C18 column, at 30°C. The mobile phase consisted of phosphate buffer solution (10 mol/L, pH 5.0): acetonitrile 60:40 (v/v) in isocratic mode, at a flow-rate of 1.0 mL/min, with only 10 min of chromatographic analyses. The mobile phase was filtered and degassed prior to use and the wavelengths used for detection were 285 and 302 nm. Plasma samples Evaluation of the HF-LPME parameters was performed using synthetic plasma consisted by the chlorides of sodium (145 mmol/L), potassium (4.5 mmol/L), calcium (32.5 mmol/L) and magnesium (0.8 mmol/L) in addition to urea (2.5 mmol/L) and glucose (4.7 mmol/L), as described in the literature (29). The synthetic plasma solution was stored at −20°C for up to 2 weeks. Plasma from healthy volunteers that had not been subjected to any pharmacological treatment for at least 72 h (blank plasma) was supplied by the Clinical Analysis Laboratory Unit of the Clinical Hospital at the Federal University of Paraná, Brazil. The plasma samples were stored at −20°C for up to 3 months and were used for validation of the method. The HF-LPME/LC-DAD method was applied to human plasma samples from six patients in PPI therapy. Written consent was obtained prior to the study. Blood samples were taken until 6 h after the final administration of the drug. It should be emphasized that the study did not interfere with the clinical conduct adopted for the patients. The study was approved by Human Research Ethics Committee of the HC at the Federal University of Paraná (Curitiba, Brazil). Methods HF-LPME apparatus and procedure All HF-LPME experiments were performed using Q3/2 Accurel polypropylene hollow fiber (600 μm i.d., 200 μm wall thickness and 0.2 μm pore size) purchased from Membrana® (Wuppertal, Germany). Before use, the hollow fiber was cleaned with acetone in an ultrasonic bath for 5 min. After drying, the fiber was cut to a length of 8 cm and then soaked in the organic solvent for 10 s to impregnate the pores. Excess solvent was removed by washing in water for 20 s in an ultrasonic bath. Subsequently, the fiber was formed into a U-shape and connected to two 25 μL liquid chromatographic microsyringes (model 702SNR—Hamilton® Reno, NV) and 25 μL of acceptor phase was injected into the lumen. Before each extraction, the microsyringes were washed 10 times with acetone and the acceptor phase, in sequence. The fiber was then immersed into the sample (donor phase) and the extraction was performed with magnetic stirring at 750 rpm (Biomixer® model 78HW-1) for 15 min at room temperature (Figure 2). Figure 2. View largeDownload slide Schematic illustration of the HF-LPME apparatus for extraction. Figure 2. View largeDownload slide Schematic illustration of the HF-LPME apparatus for extraction. For parameters experiments, the donor phase consisted of equal volumes of synthetic plasma and different buffer solutions. In a glass vial (10 mL, Shimadzu®, Japan), 100 μL of IS (20 μg/mL) and 200 μL of drug standard mix solution (100 μg/mL) were mixed, and the methanol was evaporated to dryness. The standards were then reconstituted in 5 mL of buffer solution and added to 5 mL of the synthetic plasma, resulting in a final concentration level of 2.0 and 0.2 μg/mL for the PPI drugs and IS, respectively. For analytical validation and application in real samples, the donor phase was composed by 1.0 mL of human plasma and methanol with the addition of phosphate buffer (10 mol/L, pH 5) containing 5% NaCl (w/v), to a final volume of 4.5 mL. After extraction, the microsyringes and the fiber were taken out of the sample and one of the syringes was used to collect the acceptor phase from the lumen of the fiber. The acceptor phase collected in parameters evaluation was 10 μL, which was made up to 200 μL with mobile phase and vortexed. In order to increase the enrichment factor, in validation and application experiments, the acceptor phase volume was 15 μL, with a final volume of 100 μL (with mobile phase). Finally, 50 μL of this extract was injected into the LC-DAD system. HF-LPME parameters The investigation of HF-LPME conditions was performed according to the HF-LPME procedure described. Triplicate or quadruplicate analyses were performed for all experiments and the mean values were used to plot the results. The choice of the hollow-fiber device was the first step. A low cost porous hydrophobic hollow-fiber was selected because it allows the separation of the donor and acceptor phases during the extraction procedure. In addition, as reported previously, the pore size of the fibers (0.2 μm) can block the passage of larger molecules such as plasma proteins (23, 24), decreasing interference in the chromatographic analysis caused by macromolecules. The influence of the matrix pH on the PPI extraction was investigated. For this purpose, several pH values were investigated: 5.0, 6.0 and 7.0 (phosphate buffer 10 mmol/L) for the donor phase; and pH 9.0 and 10.0 (borate buffer 10 mol/L) for the acceptor phase. The effects of the ionic strength of the matrix solution (addition of 0; 5, 10, 20 and 30% of NaCl, w/v) and the equilibrium time (10, 15, 30 and 45 min) on the PPI extraction efficiency were also investigated at room temperature. Different extraction solvents (1-octanol, butyl acetate, toluene, hexane and dichloromethane) were also evaluated for impregnation of the porous fibers. Analytical validation Validation of the HF-LPME/LC-DAD method was carried out under the selected conditions. Blank plasma samples spiked with the IS and analytes at various concentrations were used. The concentration range included the therapeutic range of the PPI drugs. Firstly, it was necessary to reduce the effect of the matrix since there was a significant binding of proteins (e.g., albumin) to the analytes. Thus, filtration in a 13-mm polycarbonate syringe filter holder (cellulose nitrate membrane 0.45 μm pores—Sartorius Stedium® Goettingen, Germany) and protein precipitation were evaluated, based on published methods in the literature (30, 31), and the solvent was changed from acetonitrile to methanol. Also, the addition of the organic modifier methanol to the donor phase to suppress protein binding without precipitation was evaluated. The selectivity of the method against endogenous interference was verified by examining the chromatograms obtained after the microextraction of blank plasma samples from at least 10 different sources. Furthermore, the selectivity of the method was investigated by comparison of the retention times among the analytes and other substances. For this purpose, the possibility of co-elution of 11 potential interfering compounds: caffeine, zidovudine, hydrochlorothiazide, diazepam, chloramphenicol, carbamazepine, acetylsalicylic acid, paracetamol, amoxicillin, diclofenac and ibuprofen was evaluated. The acceptance criterion for this study was based on the absence of substances with interfering peaks at the retention times of the drugs of interest. The linearity and limit of quantification (LOQ) were evaluated using calibration curves constructed by analyzing spiked human plasma samples after extraction by HF-LPME. This study was evaluated in triplicate, with analytes in the concentration range of LOQ, 0.50, 0.75, 1.0, 1.5 and 2.0 μg/mL. The concentration of the IS was maintained at 0.20 μg/mL. The LOQ was determined based on US-FDA recommendations (32). Accuracy, intra- and inter-day precision studies were performed by analyzing human plasma samples after HF-LPME/LC-DAD, in triplicate, with three different concentrations (low-, medium- and high-level) of PPI. For accuracy and intra-day precision, the analyte concentration levels were LOQ, 1.0 and 2.0 μg/mL while the inter-day concentrations were 0.30, 0.90 and 1.2 μg/mL. Extraction efficiency was computed similarly to the methods of Ho and coworkers (33). The enrichment factor was calculated following the methods in a previous study by Rasmussen and Perdersen-Bjergaard (25). In the study of stability, the bench stability of the analytes in the matrix at ambient temperature, the storage (freezer) stability of the plasma sample, as well as the stability of the pure standard solutions in storage and after freeze-thaw were evaluated. Results LC-DAD conditions and HF-LPME parameters The selected chromatographic conditions were a mobile phase consisting of phosphate buffer solution (10 mol/L, pH 5.0): acetonitrile 60:40 (v/v) in isocratic mode. The flow-rate was 1.0 mL/min at a temperature of 30°C, with detection at λ = 285 and 302 nm. The robustness of the method was evaluated about column temperature control (without and 30°C) and by mix standard PPI drugs solutions injections along 6 months and no significant changes were observed. The investigation of the HF-LPME parameters is shown in Figure 3 and the conditions selected were 1-octanol as solvent, phosphate buffer at pH 5 (10 mmol/L) as donor phase, borate buffer at pH 10 (10 mmol/L) as acceptor phase, extraction time of 15 min, stirring rate of 750 rpm and NaCl at 5% (w/v). Using the selected conditions, analytical validation of the HF-LPME/LC-DAD method was performed. Figure 3. View largeDownload slide Evaluation of extraction efficiency of donor and acceptor phase pH (A), extraction time (B), salt concentration addition (C) and different matrices (D) in HF-LPME method. Figure 3. View largeDownload slide Evaluation of extraction efficiency of donor and acceptor phase pH (A), extraction time (B), salt concentration addition (C) and different matrices (D) in HF-LPME method. Analytical validation First, it was necessary to study the matrix effect since protein binding may result in low recovery from human plasma samples. This effect was evaluated by comparing extraction recoveries in ultrapure water, synthetic plasma and human plasma samples (Figure 3D). Because of the plasma proteins, the application of three sample treatments was evaluated: protein filtration, protein precipitation and addition methanol and being that the latter was selected (Figure 4). Figure 4. View largeDownload slide Overlay chromatograms of HF-LPME extraction from ultrapure water (gray line), synthetic plasma (dashed line), human plasma:methanol 1:1, v/v (black line) and human plasma (dotted line). Figure 4. View largeDownload slide Overlay chromatograms of HF-LPME extraction from ultrapure water (gray line), synthetic plasma (dashed line), human plasma:methanol 1:1, v/v (black line) and human plasma (dotted line). The developed HF-LPME/LC-DAD method showed high selectivity. The retention times of the drugs of interest were different compared with those of possible interfering compounds analyzed under the same chromatography conditions in concentrations around 1 μg/mL (Table I). Furthermore, the selectivity of the method is also demonstrated by representative chromatograms of drug-free plasma after HF-LPME (blank sample) in Figure 5A, and blank samples spiked with the analytes (2.0 μg/mL) and IS (0.2 μg/mL) in Figure 5B. The chromatograms are free from interfering peaks due to endogenous compounds co-eluting with the drugs of interest. This result demonstrates the ability of the fiber to block the access of the plasma protein to the acceptor phase, as previously indicated. Table I. Retention time of analytes, IS and possible interfering compounds Compound Retention time (min) Internal standard 4.8 Omeprazole 5.9 Pantoprazole 6.7 Lansoprazole 9.0 Caffeine ND Zidovudine ND Hydrochlorothiazide ND Diazepam ND Chloramphenicol 5.0 Carbamazepine 7.3 Acetylsalycilicacid ND Paracetamol ND Amoxicillin ND Diclofenac ND Ibuprofen ND Compound Retention time (min) Internal standard 4.8 Omeprazole 5.9 Pantoprazole 6.7 Lansoprazole 9.0 Caffeine ND Zidovudine ND Hydrochlorothiazide ND Diazepam ND Chloramphenicol 5.0 Carbamazepine 7.3 Acetylsalycilicacid ND Paracetamol ND Amoxicillin ND Diclofenac ND Ibuprofen ND Compounds in bold are the analytes studied in this work. ND: not detected until 20 min of chromatographic analysis. Table I. Retention time of analytes, IS and possible interfering compounds Compound Retention time (min) Internal standard 4.8 Omeprazole 5.9 Pantoprazole 6.7 Lansoprazole 9.0 Caffeine ND Zidovudine ND Hydrochlorothiazide ND Diazepam ND Chloramphenicol 5.0 Carbamazepine 7.3 Acetylsalycilicacid ND Paracetamol ND Amoxicillin ND Diclofenac ND Ibuprofen ND Compound Retention time (min) Internal standard 4.8 Omeprazole 5.9 Pantoprazole 6.7 Lansoprazole 9.0 Caffeine ND Zidovudine ND Hydrochlorothiazide ND Diazepam ND Chloramphenicol 5.0 Carbamazepine 7.3 Acetylsalycilicacid ND Paracetamol ND Amoxicillin ND Diclofenac ND Ibuprofen ND Compounds in bold are the analytes studied in this work. ND: not detected until 20 min of chromatographic analysis. Figure 5. View largeDownload slide HF-LPME chromatograms of (A) a blank plasma sample, (B) blank plasma sample spiked with PPI drugs (2.0 μg/mL) and IS (0.2 μg/mL) and (C) patient sample in therapy with pantoprazole. Figure 5. View largeDownload slide HF-LPME chromatograms of (A) a blank plasma sample, (B) blank plasma sample spiked with PPI drugs (2.0 μg/mL) and IS (0.2 μg/mL) and (C) patient sample in therapy with pantoprazole. The linearity of the HF-LPME method was determined using drug-free plasma spiked with the PPI drugs in the range from 0.2 to 2.0 μg/mL. The analyses after HF-LPME were performed in quadruplicate. This interval was linear, with correlation coefficients >0.991 and relative standard deviations (RSD) below 15% for all concentrations in all cases (Table II). These results show that the developed method allows the determination of PPI in plasma over a wide range of concentrations. Table II. HF-LPME/LC-DAD calibration parameters Drugs Linear range Determination coefficient (R2) Intercept Slope RSD% LOQ (0.2 μg/mL) n = 3 OME 0.2–2.0 μg/mL 0.9937 0.07533 2.436 × 10−4 14.3 PAN 0.9921 0.16091 1.824 × 10−4 5.2 LANSO 0.9910 0.16239 6.356 × 10−5 9.8 Drugs Linear range Determination coefficient (R2) Intercept Slope RSD% LOQ (0.2 μg/mL) n = 3 OME 0.2–2.0 μg/mL 0.9937 0.07533 2.436 × 10−4 14.3 PAN 0.9921 0.16091 1.824 × 10−4 5.2 LANSO 0.9910 0.16239 6.356 × 10−5 9.8 Table II. HF-LPME/LC-DAD calibration parameters Drugs Linear range Determination coefficient (R2) Intercept Slope RSD% LOQ (0.2 μg/mL) n = 3 OME 0.2–2.0 μg/mL 0.9937 0.07533 2.436 × 10−4 14.3 PAN 0.9921 0.16091 1.824 × 10−4 5.2 LANSO 0.9910 0.16239 6.356 × 10−5 9.8 Drugs Linear range Determination coefficient (R2) Intercept Slope RSD% LOQ (0.2 μg/mL) n = 3 OME 0.2–2.0 μg/mL 0.9937 0.07533 2.436 × 10−4 14.3 PAN 0.9921 0.16091 1.824 × 10−4 5.2 LANSO 0.9910 0.16239 6.356 × 10−5 9.8 Moreover, the method exhibited suitable accuracy and precision. The RSD of intra-day experiments (n = 3) was <14.3% for all analytes at all three concentrations evaluated (0.2, 1.0 and 2.0 μg/mL). The accuracy at these concentrations was in the range of 87.7–108.1% and recovery values were in the range of 3.47–30.6% (Table III). Table III. HF-LPME/LC-DAD inter-day precision, accuracy, recovery, enrichment factor and intra-day precision and accuracy Drugs Inter-day Intra-day Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 Extraction efficiency (%) n = 3 Enrichment factor Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 OME 0.2 14.3 91.5 3.47 1.49 0.3 12.1 99.9 1.0 8.0 100.1 17.7 7.56 0.9 12.2 93.5 2.0 13.0 109.4 19.2 8.24 1.2 7.8 106.2 PAN 0.2 5.2 106.2 8.01 3.43 0.3 13.8 101.5 1.0 13.0 101.0 25.5 10.9 0.9 4.1 106.1 2.0 12.3 102.4 25.6 10.9 1.2 13.5 116.1 LANSO 0.2 9.8 92.6 17.6 7.35 0.3 18.2 127.3 1.0 9.1 108.1 30.6 13.1 0.9 12.9 110.4 2.0 4.4 87.7 23.6 10.1 1.2 14.4 108.8 Drugs Inter-day Intra-day Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 Extraction efficiency (%) n = 3 Enrichment factor Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 OME 0.2 14.3 91.5 3.47 1.49 0.3 12.1 99.9 1.0 8.0 100.1 17.7 7.56 0.9 12.2 93.5 2.0 13.0 109.4 19.2 8.24 1.2 7.8 106.2 PAN 0.2 5.2 106.2 8.01 3.43 0.3 13.8 101.5 1.0 13.0 101.0 25.5 10.9 0.9 4.1 106.1 2.0 12.3 102.4 25.6 10.9 1.2 13.5 116.1 LANSO 0.2 9.8 92.6 17.6 7.35 0.3 18.2 127.3 1.0 9.1 108.1 30.6 13.1 0.9 12.9 110.4 2.0 4.4 87.7 23.6 10.1 1.2 14.4 108.8 Table III. HF-LPME/LC-DAD inter-day precision, accuracy, recovery, enrichment factor and intra-day precision and accuracy Drugs Inter-day Intra-day Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 Extraction efficiency (%) n = 3 Enrichment factor Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 OME 0.2 14.3 91.5 3.47 1.49 0.3 12.1 99.9 1.0 8.0 100.1 17.7 7.56 0.9 12.2 93.5 2.0 13.0 109.4 19.2 8.24 1.2 7.8 106.2 PAN 0.2 5.2 106.2 8.01 3.43 0.3 13.8 101.5 1.0 13.0 101.0 25.5 10.9 0.9 4.1 106.1 2.0 12.3 102.4 25.6 10.9 1.2 13.5 116.1 LANSO 0.2 9.8 92.6 17.6 7.35 0.3 18.2 127.3 1.0 9.1 108.1 30.6 13.1 0.9 12.9 110.4 2.0 4.4 87.7 23.6 10.1 1.2 14.4 108.8 Drugs Inter-day Intra-day Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 Extraction efficiency (%) n = 3 Enrichment factor Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 OME 0.2 14.3 91.5 3.47 1.49 0.3 12.1 99.9 1.0 8.0 100.1 17.7 7.56 0.9 12.2 93.5 2.0 13.0 109.4 19.2 8.24 1.2 7.8 106.2 PAN 0.2 5.2 106.2 8.01 3.43 0.3 13.8 101.5 1.0 13.0 101.0 25.5 10.9 0.9 4.1 106.1 2.0 12.3 102.4 25.6 10.9 1.2 13.5 116.1 LANSO 0.2 9.8 92.6 17.6 7.35 0.3 18.2 127.3 1.0 9.1 108.1 30.6 13.1 0.9 12.9 110.4 2.0 4.4 87.7 23.6 10.1 1.2 14.4 108.8 In the inter-day assay, the RSD (n = 3) values were <13.8% for all concentrations (0.3, 0.9 and 1.2 μg/mL) for OME and PAN, while for LANSO the RSD was 18.2%. However, as discussed above, LANSO is the analyte with the lowest stability in acid conditions and, as consequence, showed lower sensitivity in the calibration curve. Considering the measured accuracy, the inter-day experiments were between 93.5 and 106.2% for OME and PAN and from 108.8% to 127.3% for LANSO (Table III). The stability of the plasma samples spiked with drugs at different concentrations (0.2, 1.0 and 2.0 μg/mL) was monitored for 8 h (the time required for sample preparation and analysis in a single day) at ambient temperature, 25°C. No statistically significant differences were observed for the three different concentrations. The patient plasma samples were stored at −20°C for 2 weeks and these samples were stable for longer time periods under these conditions. The plasma samples spiked with PPI drugs at concentrations between 0.2 and 1.0 μg/mL were stable after three freeze-thaw cycles (in each freeze/thaw cycle, the samples were frozen at −20°C for 24 h, thawed and then maintained at ambient temperature for 1 h). The working solutions kept at −18°C and protected from light were stable for over 12 months. Method application In order to evaluate the proposed HF-LPME/LC-DAD method for clinical use, the described protocol was applied to the analysis of plasma samples from patients in therapy with the studied PPI drugs. The peak shapes and resolution were very similar to those obtained using spiked blank plasma and no interference was apparent. These plasma samples were collected from patients in therapy with OME (20 mg/day) and PAN sodium sesquihydrate (20 mg/day), respectively. The drug concentrations found in these samples were 1.12 and 1.15 μg/mL for OME (data not shown) and 1.92 μg/mL for PAN (Figure 5C). The plasma concentration values are within the linear range of the method, which was considered suitable based on a maximal plasma drug concentration between 0.2 and 2.0 μg/mL. Discussion LC-DAD conditions Several different elution conditions were evaluated for the simultaneous separation of the PPI drugs on the C18 column in order to obtain good resolution with a short analysis time. Initially, acetonitrile-water was used as the mobile phase, however, peak broadening was observed under these conditions. Thus, different mobile phase compositions incorporating phosphate (10 mol/L, pH at 3.0; 5.0; 6.0 and 7.0) and borate buffer solutions (10 mol/L, pH at 9.0) and acetonitrile were investigated, varying the acetonitrile ratio between 50 and 65% (v/v). Under alkaline conditions (pH 9.0) the analytes are predominantly in anionic form, while at pH 3.0 they are mostly cationic. Under these elution conditions, the retention times of these ionic forms of the drugs were close to each other and to the column dead volume, evidencing low selectivity. Due to the pKa values of the analytes, neutral forms are in the majority in the pH range 5.0–7.0. Thus, mobile phase compositions with acetonitrile and phosphate buffer solutions (10 mol/L) in this pH range were explored. Although asymmetric peaks were observed, improved resolution was achieved with phosphate buffer solution at pH 5.0 and acetonitrile (60:40 v/v) with a short 10-min chromatographic run. The addition of triethylamine (0.01%) in the mobile phase was tested, but no improvement in the peak symmetry was observed. As discussed by Santos Neto (34), peak distortions may occur when the solvent sample has a higher elution strength than the mobile phase. The PPI mix working solution was prepared in methanol, which has a higher elution strength than the combination of pH 5.0 phosphate buffer solution and acetonitrile (60:40 v/v). Hence, an improvement in the peak shape of the analytes was achieved by drying the methanol-PPI sample and redissolving it in the selected mobile phase. Based on literature, the wavelengths of 270, 285, 294 and 302 nm were investigated for evaluation of the maximum absorption of the analytes (2, 35, 36). It was confirmed that the λmax for OME was at 302 nm, while the maximum absorption for PAN and LANSO was at 285 nm (Figure 6). The IS λmax was at 270 nm, with significant absorption at 285 nm. Hence, for study of the HF-LPME parameters, 285 nm was selected for all drugs, although for validation and application in real samples the wavelength 302 nm was preferred for OME in order to improve the peak intensity (better detectability). Figure 6. View largeDownload slide Spectral UV of PPI drugs omeprazole, pantoprazole and lansoprazole with the wavelength selected. Figure 6. View largeDownload slide Spectral UV of PPI drugs omeprazole, pantoprazole and lansoprazole with the wavelength selected. Evaluation of the HF-LPME parameters Selection of the organic solvent is a crucial parameter as it is the extractor phase in both HF-LPME extraction modes. A suitable solvent should have low solubility in water to prevent leakage into the aqueous solution and maintain stability at the pores of the hydrophobic fiber. It is also advantageous to have low volatility in the three-phase mode, to avoid evaporation during extraction (24, 37). Five organic solvents that are commonly used in the two and three-phase methods were evaluated in triplicate: hexane, toluene, 1-octanol, dichloromethane and butyl acetate (25). Of these, butyl acetate and 1-octanol met the requirements for low volatility and low solubility in water for the three-phase mode process. When toluene, dichloromethane and hexane were used, solvent losses were observed during sample preparation and it was not possible to collect reproducible volumes of the acceptor phase. Between butyl acetate and 1-octanol, only 1-octanol satisfactorily extracted the PPI drugs and IS in three-phase mode in this study. Because an alkaline aqueous acceptor phase and an acidic donor phase were employed, butyl acetate hydrolysis may have occurred in one of these environments, as discussed in the literature (38, 39). Thus, 1-octanol was selected as the supported organic solvent. The pH of the donor phase (sample solution) was adjusted to be in the range 5.0–7.0 to maintain the analytes predominantly at neutral charge and improve the mass transfer into the organic solvent. Lower pH values cause the analytes to be in cationic form and as a consequence they may become degraded, since the PPI drugs are acid labeled. Therefore, the pH of the donor phase was adjusted with phosphate buffer solutions (10 mol/L) of pH 5.0, 6.0 and 7.0. The highest extraction recoveries were obtained with the donor phase at pH 5.0 or 6.0, (the difference between them not being significant) (Figure 3A); while a decrease in recovery was detected at pH 7.0. This might be attributed to deprotonation occurring at higher pH, causing ionization of the drugs. As the mobile phase of the chromatographic method was composed by phosphate buffer at pH 5.0 and acetonitrile, pH 5.0 phosphate buffer solution was selected for the donor phase to facilitate injection of the sample into the analytical system. The pH of the aqueous acceptor phase used in three-phase mode is modified to ensure that the analyte molecules are predominantly in the anionic form, and thus prevent them from re-entering the organic phase (24). Borate buffer solutions (10 mol/L) at pH 9.0 and 10.0 were evaluated for this purpose. With a pH 9.0 acceptor phase, a lower extraction recovery was observed for all drugs, compared with pH 10.0 (Figure 3A). This behavior might be attributed to the fact that the ionized forms of the PPI drugs are more favored when the pH is increased. Accordingly, the selected acceptor phase was borate solution (10 mol/L) at pH 10. Using the selected organic solvent and the optimum pH conditions for the donor and acceptor phases, the influence of the extraction time was evaluated in the range of 10–45 min. Distinct behaviors were observed for the different analytes, which may be associated with their physical and chemical differences based on the nature of the substituents on the PPI drug molecules (7). As a result (Figure 3B), it was observed that PAN reached partition equilibrium after 30 min of extraction. OME also reached mass transfer equilibrium at this extraction time, however, there was a decrease in the peak area beginning at 15 min. Finally, LANSO did not reach a plateau in the tested time period and its RSD values increased from 17% at 10 min to 54% at 45 min. Considering these results, an extraction time of 15 min was selected. These results show that it is possible to employ the HF-LPME technique under non-equilibrium conditions for PPI drug extraction in the three-phase mode, despite their instability under acid conditions. Magnetic stirring improves the mass transfer of analytes from the plasma sample to the hollow-fiber. It is known that for solid-phase microextraction, the effect of the static layer zone close to the fiber is reduced by stirring (40). Similarly, for the hollow-fiber in the present study, the samples were stirred during extraction at a maximum rate of 750 rpm, without loss of extraction solvent from the fiber. The possibility of salting out effect was also investigated by increasing the NaCl concentration between 0 and 30% (w/v) in the HF-LPME extraction of the PPI drugs. As shown in Figure 3C, there was a rise in the extraction efficiency with increasing salt concentration up to 5% and a reduction >10%. The addition of salt may decrease analyte solubility since the water molecules of the sample hydrate ionic salt molecules and the reduction in extraction efficiency is explained by the fact that electrostatic interactions can occur between polar molecules and salt ions (41). In addition, increased salt concentration may increase the donor-phase viscosity and lead to emulsion formation. Based on this, a salt concentration of 5% (w/v) was selected. Analytical validation As shown in Figure 3D, human plasma samples showed decreased extraction efficiency, indicating an effect of matrix compounds, mainly plasma proteins. As a result, the application of three sample treatments was evaluated: protein filtration, protein precipitation and addition of the organic modifier methanol. Methanol was tested as when lower recoveries are observed in plasma than in water, the main interactions might be hydrophobic and the addition of an organic modifier such as methanol can suppress this binding (33). Precipitation or filtration did not improve the extraction efficiency. This may be because of drug–protein binding the analytes may be precipitated and filtered out along with the proteins. On the other hand, the addition of methanol showed an improvement in extraction recovery at a human plasma:methanol ratio of 1:1 (v/v) and decreases with higher proportions of methanol. The addition of methanol may affect the distribution of the analyte in the aqueous and organic phases, decreasing the extraction recovery (42). Although the addition of methanol to plasma did not improve the extraction recovery to a level comparable with that in ultrapure water or synthetic plasma, a significant increase in recovery was achieved compared with plasma samples without treatment, as shown in Figure 4. Since the RSD at the lowest concentration level (0.2 μg/mL) was <20% and showed accuracy in the range of 80–120%, this could be considered as the lower LOQ according to US-FDA recommendations. The LOQ is higher than in literature references concerning PPI drug determination, which range from 2.5 to 20 ng/mL. However, as previously mentioned, these drugs are typically extracted using conventional exhaustive techniques such as LLE and SPE (17, 43, 44) and, in some cases, with calibration curves measured in solvent or in the mobile phase. Also, highly sensitive analytical techniques such as LC-MS and LC-MS/MS are commonly used (16, 45). Beyond that, it should be noted that there are no documented studies employing HF-LPME for PPI drug determination in human plasma in a simple analytical system such as LC-DAD. Furthermore, the maximal plasma concentrations of the target drugs range between 0.5 and 1.5 μg/mL in the interval of 1–4 h (46, 47). Based on this information, an analytical method should be able to determinate these drugs in the concentration range 0.2–2.0 μg/mL assuming sampling at the time of maximal plasma concentration, the LOQ obtained in this paper is in this concentration range. Extraction efficiency values were satisfactory, in the range of 3.5–30%, as was the enrichment factor (between 1.5 and 13), considering the significant matrix effect. It can also be highlighted that HF-LPME is not an exhaustive but an equilibrium technique (42), however, the extraction should be performed rapidly as a consequence of the acid instability of the analytes. Recoveries with SPE or LLE techniques of these drugs are between 50 and 110% (17, 18, 45, 47). The developed method and sample preparation can be performed under non-equilibrium conditions with a mere 15 min of extraction time, while achieving the proper accuracy and precision. Furthermore, studies employing HF-LPME typically report recoveries near to 10% for biological samples (48). Moreover, there are also reports in the literature with recoveries <10%, for example, absolute recoveries in the range of 4.4–8.9% for the determination of cannabinoids in human hair by GC-MS/MS (49). Method application This application of the method shows that is possible to employ the HF-LPME microextraction technique for PPI drug determination in plasma samples, contributing to studies of the adverse effects of these drugs in long-term therapy. Conclusions In this study, an HF-LPME/LC-DAD method was developed, validated and applied for the determination of OME, PAN and LANSO in human plasma samples. This allowed a low cost, rapid, sensitive, selective and reproducible methodology for the determination of these drugs. In addition, the requirement for organic solvents is reduced and the sample volume used is lower than in conventional techniques, which is important for invasive samples such as human plasma. Although the matrix complexity, it was possible to apply the HF-LPME technique with reduced matrix effects, in fast analyses, and obtain clean extracts without interferents, because the pore size of the fiber (0.2-μm) can block the passage of larger molecules such as plasma proteins and decrease the interference of macromolecules in chromatographic analysis. The sample preparation could also be performed under non-equilibrium conditions with suitable accuracy and precision. The investigation of the HF-LPME parameters was crucial to allow the determination of PPI drugs in acid conditions. This study contributes to knowledge about the determination of these types of drugs using miniaturized techniques and will enable studies concerning adverse drug effects. Finally, the HF-LPME/LC-DAD method was successfully applied in human plasma samples from patients undergoing therapy with OME and PAN. 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Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Evaluation of a Hollow-Fiber Liquid-Phase Microextraction Technique for the Simultaneous Determination of PPI Drugs in Human Plasma by LC-DAD

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Abstract

Abstract This study involved the development, validation and application of a three-phase hollow-fiber liquid-phase microextraction (HF-LPME) and liquid chromatography with diode array detection (LC-DAD) method for the simultaneous determination of the proton pump inhibitor (PPI) drugs omeprazole, pantoprazole and lansoprazole in human plasma. The evaluation of the HF-LPME parameters was crucial for the determination of the drugs and the conditions selected were: 1-octanol as solvent; phosphate buffer at pH 5 as donor phase; borate buffer at pH 10 as acceptor phase; extraction time of 15 min; stirring at 750 rpm and NaCl was added at 5% (w/v). Validation of the method according to US-FDA recommendations showed a good linear range (0.2–2.0 μg/mL) for all analytes, with a determination coefficient >0.9910. Precision was evaluated using intra- and inter-day assays, which showed relative standard deviations (RSD), <15% for all concentrations, with a limit of quantification (LOQ) of 0.2 μg/mL. Accuracy was also assessed at these concentration levels and was in the range from 80 to 130%. Finally, the sensitive, selective and reproducible HF-LPME/LC-DAD developed method was successfully applied to human plasma samples from patients undergoing therapy with the PPI drugs. Introduction Proton pump inhibitors (PPI) are some of the most widely prescribed drugs used in the treatment of disorders related to gastric acid secretion (1). PPIs, which include omeprazole (OME), pantoprazole (PAN) and lansoprazole (LANSO), are weak basic prodrugs that are converted to their active form in acid conditions. These activated forms block the gastric H+/K+ATPase (proton pump enzyme) which is responsible for the final step of the acid secretion process (2–4). As they have pKa1 and pKa2 values around 4 and 9, respectively (1), their activation can occur in the acid conditions of the stomach parietal cells (pH 1.3) that is their site of action (5). This is reflected in the fact that this class of drugs is more effective than others in the suppression of acid secretion (6). OME, PAN and LANSO have the same general mechanism of action and are unstable in acid environments but their different substituents on the pyridine and benzimidazole groups (Figure 1) result in some distinct properties (7). Concerning their acid stability, for example, the degradation rate is different and the stability order is: PAN > OME > LANSO (3). Another pharmacokinetic difference is the time taken to reach peak plasma concentration that is in the range of 0.5–3.5 h for OME, 1.7 h for LANSO and 1.1–3.1 h for PAN (8). All PPIs are rapidly absorbed and are highly plasma protein bound, which results in a low volume of distribution (9). The proportion of the drug bound to protein is 95% for OME, 98% for LANSO and 97% for PAN (8, 10, 11). Figure 1. View largeDownload slide Chemical structure of selected PPI drugs. Figure 1. View largeDownload slide Chemical structure of selected PPI drugs. Recent studies have made an association between chronic PPI therapy and potential adverse effects such as dementia, an increased risk of bone fractures and deficiency of vitamin B12 among others (12–15). For this reason, it is important to develop new methodologies to determine these drugs in biological matrices in order to evaluate their effects. In the literature, there are few works where the simultaneous determination of PPI drugs in human plasma has been performed, as reported by Bharathi and coworkers (2). Most studies concern one PPI drug and its enantiomers with their respective metabolites. They are usually determined in human plasma using liquid–liquid extraction (LLE) (2, 16, 17) or solid-phase extraction (SPE) (18) with liquid chromatography, usually coupled with tandem mass spectrometry (LC-MS/MS) or ultraviolet detection (LC-UV or LC-DAD). However, these conventional extraction methods have some disadvantages, such as the consumption of large volumes of toxic organic solvents, the requirement of costly cartridges and they are time-consuming (19–21). The hollow-fiber liquid-phase microextraction (HF-LPME) technique has received some attention since it uses low volumes of organic solvents to extract organic compounds from small amounts of aqueous samples (22). Furthermore, the technique employs a porous hydrophobic hollow fiber. This inexpensive disposable device eliminates the carry-over effect and its pore size can block the passage of larger molecules such as plasma proteins (23, 24). HF-LPME was introduced in 1999 (24) and performs the extraction of compounds from a donor phase (sample) through an organic solvent (extractor phase) into an acceptor phase inside the lumen of a hollow fiber. This acceptor phase can be the same extractor organic solvent as that immobilized in the fiber pores, corresponding to the two-phase mode; alternatively, it can be an aqueous solution when the technique is used in the three-phase mode (20). Because the extractor and acceptor phases are, respectively, immobilized in the pores and inside the lumen, the sample can be stirred or vibrated to accelerate the reaction kinetics without the loss of the phases (25, 26). There have been several reports of HF-LPME applications for drug and metabolite extraction from biological matrices (19, 27, 28). Concerning PPI drugs, to our knowledge, there is no documented HF-LPME method for the simultaneous determination of OME, PAN and LANSO in human plasma samples. Differently to previous studies concerning the determination of PPI drugs, in this paper we developed a miniaturized method (HF-LPME) for the simultaneous determination of three PPI drugs in human plasma by LC-DAD: OME, PAN and LANSO. The chromatographic and extraction parameters were evaluated in order to enable the extraction of these acid labeled and highly protein bound drugs from this complex matrix. Moreover, the method was validated and applied to human plasma samples from patients undergoing PPI therapy. Experimental Reagents OME, PAN, LANSO and sulfamethoxazole (internal standard, IS) analytical standards were purchased from Fluka Sigma-Aldrich® (São Paulo, Brazil). Monobasic potassium phosphate and high performance liquid chromatography (HPLC) grade acetonitrile and methanol were obtained from J.T. Baker® (Phillipsburg, USA). Dibasic potassium phosphate (Vetec®, Rio de Janeiro, Brazil) and boric acid (Isofar®, Rio de Janeiro, Brazil) were used to prepare buffer solutions in ultrapure water purified by a Milli-Q Millipore® system (18 MΩ at 25°C) (São Paulo, Brazil). Working standard drug solutions were prepared by the diluting 1 mg/mL methanol stock solutions to the appropriate volume with methanol and kept at −18°C protected from light. Hexane, toluene and 1-octanol were purchased from Sigma-Aldrich® (São Paulo, Brazil). Butyl acetate (F. Maia®, São Paulo, Brazil) and dichloromethane (Vetec®, Rio de Janeiro, Brazil) were also evaluated as organic solvents. pH adjustments were made using diluted solutions of sodium hydroxide (Proquímios®, Rio de Janeiro, Brazil) and hydrochloric acid (Dinâmica®, São Paulo, Brazil). Reagents used for synthetic plasma were all of analytical grade. Instrumentation and chromatographic conditions Chromatographic analyses were performed using an Agilent® 1260 Infinity HPLC system with quaternary pump and diode array detection. The separation was achieved using an Agilent® Microsorb MV100-5 C18 column (250 mm × 4.6 mm, 5 μm), preceded by an Agilent® guard C18 column, at 30°C. The mobile phase consisted of phosphate buffer solution (10 mol/L, pH 5.0): acetonitrile 60:40 (v/v) in isocratic mode, at a flow-rate of 1.0 mL/min, with only 10 min of chromatographic analyses. The mobile phase was filtered and degassed prior to use and the wavelengths used for detection were 285 and 302 nm. Plasma samples Evaluation of the HF-LPME parameters was performed using synthetic plasma consisted by the chlorides of sodium (145 mmol/L), potassium (4.5 mmol/L), calcium (32.5 mmol/L) and magnesium (0.8 mmol/L) in addition to urea (2.5 mmol/L) and glucose (4.7 mmol/L), as described in the literature (29). The synthetic plasma solution was stored at −20°C for up to 2 weeks. Plasma from healthy volunteers that had not been subjected to any pharmacological treatment for at least 72 h (blank plasma) was supplied by the Clinical Analysis Laboratory Unit of the Clinical Hospital at the Federal University of Paraná, Brazil. The plasma samples were stored at −20°C for up to 3 months and were used for validation of the method. The HF-LPME/LC-DAD method was applied to human plasma samples from six patients in PPI therapy. Written consent was obtained prior to the study. Blood samples were taken until 6 h after the final administration of the drug. It should be emphasized that the study did not interfere with the clinical conduct adopted for the patients. The study was approved by Human Research Ethics Committee of the HC at the Federal University of Paraná (Curitiba, Brazil). Methods HF-LPME apparatus and procedure All HF-LPME experiments were performed using Q3/2 Accurel polypropylene hollow fiber (600 μm i.d., 200 μm wall thickness and 0.2 μm pore size) purchased from Membrana® (Wuppertal, Germany). Before use, the hollow fiber was cleaned with acetone in an ultrasonic bath for 5 min. After drying, the fiber was cut to a length of 8 cm and then soaked in the organic solvent for 10 s to impregnate the pores. Excess solvent was removed by washing in water for 20 s in an ultrasonic bath. Subsequently, the fiber was formed into a U-shape and connected to two 25 μL liquid chromatographic microsyringes (model 702SNR—Hamilton® Reno, NV) and 25 μL of acceptor phase was injected into the lumen. Before each extraction, the microsyringes were washed 10 times with acetone and the acceptor phase, in sequence. The fiber was then immersed into the sample (donor phase) and the extraction was performed with magnetic stirring at 750 rpm (Biomixer® model 78HW-1) for 15 min at room temperature (Figure 2). Figure 2. View largeDownload slide Schematic illustration of the HF-LPME apparatus for extraction. Figure 2. View largeDownload slide Schematic illustration of the HF-LPME apparatus for extraction. For parameters experiments, the donor phase consisted of equal volumes of synthetic plasma and different buffer solutions. In a glass vial (10 mL, Shimadzu®, Japan), 100 μL of IS (20 μg/mL) and 200 μL of drug standard mix solution (100 μg/mL) were mixed, and the methanol was evaporated to dryness. The standards were then reconstituted in 5 mL of buffer solution and added to 5 mL of the synthetic plasma, resulting in a final concentration level of 2.0 and 0.2 μg/mL for the PPI drugs and IS, respectively. For analytical validation and application in real samples, the donor phase was composed by 1.0 mL of human plasma and methanol with the addition of phosphate buffer (10 mol/L, pH 5) containing 5% NaCl (w/v), to a final volume of 4.5 mL. After extraction, the microsyringes and the fiber were taken out of the sample and one of the syringes was used to collect the acceptor phase from the lumen of the fiber. The acceptor phase collected in parameters evaluation was 10 μL, which was made up to 200 μL with mobile phase and vortexed. In order to increase the enrichment factor, in validation and application experiments, the acceptor phase volume was 15 μL, with a final volume of 100 μL (with mobile phase). Finally, 50 μL of this extract was injected into the LC-DAD system. HF-LPME parameters The investigation of HF-LPME conditions was performed according to the HF-LPME procedure described. Triplicate or quadruplicate analyses were performed for all experiments and the mean values were used to plot the results. The choice of the hollow-fiber device was the first step. A low cost porous hydrophobic hollow-fiber was selected because it allows the separation of the donor and acceptor phases during the extraction procedure. In addition, as reported previously, the pore size of the fibers (0.2 μm) can block the passage of larger molecules such as plasma proteins (23, 24), decreasing interference in the chromatographic analysis caused by macromolecules. The influence of the matrix pH on the PPI extraction was investigated. For this purpose, several pH values were investigated: 5.0, 6.0 and 7.0 (phosphate buffer 10 mmol/L) for the donor phase; and pH 9.0 and 10.0 (borate buffer 10 mol/L) for the acceptor phase. The effects of the ionic strength of the matrix solution (addition of 0; 5, 10, 20 and 30% of NaCl, w/v) and the equilibrium time (10, 15, 30 and 45 min) on the PPI extraction efficiency were also investigated at room temperature. Different extraction solvents (1-octanol, butyl acetate, toluene, hexane and dichloromethane) were also evaluated for impregnation of the porous fibers. Analytical validation Validation of the HF-LPME/LC-DAD method was carried out under the selected conditions. Blank plasma samples spiked with the IS and analytes at various concentrations were used. The concentration range included the therapeutic range of the PPI drugs. Firstly, it was necessary to reduce the effect of the matrix since there was a significant binding of proteins (e.g., albumin) to the analytes. Thus, filtration in a 13-mm polycarbonate syringe filter holder (cellulose nitrate membrane 0.45 μm pores—Sartorius Stedium® Goettingen, Germany) and protein precipitation were evaluated, based on published methods in the literature (30, 31), and the solvent was changed from acetonitrile to methanol. Also, the addition of the organic modifier methanol to the donor phase to suppress protein binding without precipitation was evaluated. The selectivity of the method against endogenous interference was verified by examining the chromatograms obtained after the microextraction of blank plasma samples from at least 10 different sources. Furthermore, the selectivity of the method was investigated by comparison of the retention times among the analytes and other substances. For this purpose, the possibility of co-elution of 11 potential interfering compounds: caffeine, zidovudine, hydrochlorothiazide, diazepam, chloramphenicol, carbamazepine, acetylsalicylic acid, paracetamol, amoxicillin, diclofenac and ibuprofen was evaluated. The acceptance criterion for this study was based on the absence of substances with interfering peaks at the retention times of the drugs of interest. The linearity and limit of quantification (LOQ) were evaluated using calibration curves constructed by analyzing spiked human plasma samples after extraction by HF-LPME. This study was evaluated in triplicate, with analytes in the concentration range of LOQ, 0.50, 0.75, 1.0, 1.5 and 2.0 μg/mL. The concentration of the IS was maintained at 0.20 μg/mL. The LOQ was determined based on US-FDA recommendations (32). Accuracy, intra- and inter-day precision studies were performed by analyzing human plasma samples after HF-LPME/LC-DAD, in triplicate, with three different concentrations (low-, medium- and high-level) of PPI. For accuracy and intra-day precision, the analyte concentration levels were LOQ, 1.0 and 2.0 μg/mL while the inter-day concentrations were 0.30, 0.90 and 1.2 μg/mL. Extraction efficiency was computed similarly to the methods of Ho and coworkers (33). The enrichment factor was calculated following the methods in a previous study by Rasmussen and Perdersen-Bjergaard (25). In the study of stability, the bench stability of the analytes in the matrix at ambient temperature, the storage (freezer) stability of the plasma sample, as well as the stability of the pure standard solutions in storage and after freeze-thaw were evaluated. Results LC-DAD conditions and HF-LPME parameters The selected chromatographic conditions were a mobile phase consisting of phosphate buffer solution (10 mol/L, pH 5.0): acetonitrile 60:40 (v/v) in isocratic mode. The flow-rate was 1.0 mL/min at a temperature of 30°C, with detection at λ = 285 and 302 nm. The robustness of the method was evaluated about column temperature control (without and 30°C) and by mix standard PPI drugs solutions injections along 6 months and no significant changes were observed. The investigation of the HF-LPME parameters is shown in Figure 3 and the conditions selected were 1-octanol as solvent, phosphate buffer at pH 5 (10 mmol/L) as donor phase, borate buffer at pH 10 (10 mmol/L) as acceptor phase, extraction time of 15 min, stirring rate of 750 rpm and NaCl at 5% (w/v). Using the selected conditions, analytical validation of the HF-LPME/LC-DAD method was performed. Figure 3. View largeDownload slide Evaluation of extraction efficiency of donor and acceptor phase pH (A), extraction time (B), salt concentration addition (C) and different matrices (D) in HF-LPME method. Figure 3. View largeDownload slide Evaluation of extraction efficiency of donor and acceptor phase pH (A), extraction time (B), salt concentration addition (C) and different matrices (D) in HF-LPME method. Analytical validation First, it was necessary to study the matrix effect since protein binding may result in low recovery from human plasma samples. This effect was evaluated by comparing extraction recoveries in ultrapure water, synthetic plasma and human plasma samples (Figure 3D). Because of the plasma proteins, the application of three sample treatments was evaluated: protein filtration, protein precipitation and addition methanol and being that the latter was selected (Figure 4). Figure 4. View largeDownload slide Overlay chromatograms of HF-LPME extraction from ultrapure water (gray line), synthetic plasma (dashed line), human plasma:methanol 1:1, v/v (black line) and human plasma (dotted line). Figure 4. View largeDownload slide Overlay chromatograms of HF-LPME extraction from ultrapure water (gray line), synthetic plasma (dashed line), human plasma:methanol 1:1, v/v (black line) and human plasma (dotted line). The developed HF-LPME/LC-DAD method showed high selectivity. The retention times of the drugs of interest were different compared with those of possible interfering compounds analyzed under the same chromatography conditions in concentrations around 1 μg/mL (Table I). Furthermore, the selectivity of the method is also demonstrated by representative chromatograms of drug-free plasma after HF-LPME (blank sample) in Figure 5A, and blank samples spiked with the analytes (2.0 μg/mL) and IS (0.2 μg/mL) in Figure 5B. The chromatograms are free from interfering peaks due to endogenous compounds co-eluting with the drugs of interest. This result demonstrates the ability of the fiber to block the access of the plasma protein to the acceptor phase, as previously indicated. Table I. Retention time of analytes, IS and possible interfering compounds Compound Retention time (min) Internal standard 4.8 Omeprazole 5.9 Pantoprazole 6.7 Lansoprazole 9.0 Caffeine ND Zidovudine ND Hydrochlorothiazide ND Diazepam ND Chloramphenicol 5.0 Carbamazepine 7.3 Acetylsalycilicacid ND Paracetamol ND Amoxicillin ND Diclofenac ND Ibuprofen ND Compound Retention time (min) Internal standard 4.8 Omeprazole 5.9 Pantoprazole 6.7 Lansoprazole 9.0 Caffeine ND Zidovudine ND Hydrochlorothiazide ND Diazepam ND Chloramphenicol 5.0 Carbamazepine 7.3 Acetylsalycilicacid ND Paracetamol ND Amoxicillin ND Diclofenac ND Ibuprofen ND Compounds in bold are the analytes studied in this work. ND: not detected until 20 min of chromatographic analysis. Table I. Retention time of analytes, IS and possible interfering compounds Compound Retention time (min) Internal standard 4.8 Omeprazole 5.9 Pantoprazole 6.7 Lansoprazole 9.0 Caffeine ND Zidovudine ND Hydrochlorothiazide ND Diazepam ND Chloramphenicol 5.0 Carbamazepine 7.3 Acetylsalycilicacid ND Paracetamol ND Amoxicillin ND Diclofenac ND Ibuprofen ND Compound Retention time (min) Internal standard 4.8 Omeprazole 5.9 Pantoprazole 6.7 Lansoprazole 9.0 Caffeine ND Zidovudine ND Hydrochlorothiazide ND Diazepam ND Chloramphenicol 5.0 Carbamazepine 7.3 Acetylsalycilicacid ND Paracetamol ND Amoxicillin ND Diclofenac ND Ibuprofen ND Compounds in bold are the analytes studied in this work. ND: not detected until 20 min of chromatographic analysis. Figure 5. View largeDownload slide HF-LPME chromatograms of (A) a blank plasma sample, (B) blank plasma sample spiked with PPI drugs (2.0 μg/mL) and IS (0.2 μg/mL) and (C) patient sample in therapy with pantoprazole. Figure 5. View largeDownload slide HF-LPME chromatograms of (A) a blank plasma sample, (B) blank plasma sample spiked with PPI drugs (2.0 μg/mL) and IS (0.2 μg/mL) and (C) patient sample in therapy with pantoprazole. The linearity of the HF-LPME method was determined using drug-free plasma spiked with the PPI drugs in the range from 0.2 to 2.0 μg/mL. The analyses after HF-LPME were performed in quadruplicate. This interval was linear, with correlation coefficients >0.991 and relative standard deviations (RSD) below 15% for all concentrations in all cases (Table II). These results show that the developed method allows the determination of PPI in plasma over a wide range of concentrations. Table II. HF-LPME/LC-DAD calibration parameters Drugs Linear range Determination coefficient (R2) Intercept Slope RSD% LOQ (0.2 μg/mL) n = 3 OME 0.2–2.0 μg/mL 0.9937 0.07533 2.436 × 10−4 14.3 PAN 0.9921 0.16091 1.824 × 10−4 5.2 LANSO 0.9910 0.16239 6.356 × 10−5 9.8 Drugs Linear range Determination coefficient (R2) Intercept Slope RSD% LOQ (0.2 μg/mL) n = 3 OME 0.2–2.0 μg/mL 0.9937 0.07533 2.436 × 10−4 14.3 PAN 0.9921 0.16091 1.824 × 10−4 5.2 LANSO 0.9910 0.16239 6.356 × 10−5 9.8 Table II. HF-LPME/LC-DAD calibration parameters Drugs Linear range Determination coefficient (R2) Intercept Slope RSD% LOQ (0.2 μg/mL) n = 3 OME 0.2–2.0 μg/mL 0.9937 0.07533 2.436 × 10−4 14.3 PAN 0.9921 0.16091 1.824 × 10−4 5.2 LANSO 0.9910 0.16239 6.356 × 10−5 9.8 Drugs Linear range Determination coefficient (R2) Intercept Slope RSD% LOQ (0.2 μg/mL) n = 3 OME 0.2–2.0 μg/mL 0.9937 0.07533 2.436 × 10−4 14.3 PAN 0.9921 0.16091 1.824 × 10−4 5.2 LANSO 0.9910 0.16239 6.356 × 10−5 9.8 Moreover, the method exhibited suitable accuracy and precision. The RSD of intra-day experiments (n = 3) was <14.3% for all analytes at all three concentrations evaluated (0.2, 1.0 and 2.0 μg/mL). The accuracy at these concentrations was in the range of 87.7–108.1% and recovery values were in the range of 3.47–30.6% (Table III). Table III. HF-LPME/LC-DAD inter-day precision, accuracy, recovery, enrichment factor and intra-day precision and accuracy Drugs Inter-day Intra-day Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 Extraction efficiency (%) n = 3 Enrichment factor Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 OME 0.2 14.3 91.5 3.47 1.49 0.3 12.1 99.9 1.0 8.0 100.1 17.7 7.56 0.9 12.2 93.5 2.0 13.0 109.4 19.2 8.24 1.2 7.8 106.2 PAN 0.2 5.2 106.2 8.01 3.43 0.3 13.8 101.5 1.0 13.0 101.0 25.5 10.9 0.9 4.1 106.1 2.0 12.3 102.4 25.6 10.9 1.2 13.5 116.1 LANSO 0.2 9.8 92.6 17.6 7.35 0.3 18.2 127.3 1.0 9.1 108.1 30.6 13.1 0.9 12.9 110.4 2.0 4.4 87.7 23.6 10.1 1.2 14.4 108.8 Drugs Inter-day Intra-day Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 Extraction efficiency (%) n = 3 Enrichment factor Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 OME 0.2 14.3 91.5 3.47 1.49 0.3 12.1 99.9 1.0 8.0 100.1 17.7 7.56 0.9 12.2 93.5 2.0 13.0 109.4 19.2 8.24 1.2 7.8 106.2 PAN 0.2 5.2 106.2 8.01 3.43 0.3 13.8 101.5 1.0 13.0 101.0 25.5 10.9 0.9 4.1 106.1 2.0 12.3 102.4 25.6 10.9 1.2 13.5 116.1 LANSO 0.2 9.8 92.6 17.6 7.35 0.3 18.2 127.3 1.0 9.1 108.1 30.6 13.1 0.9 12.9 110.4 2.0 4.4 87.7 23.6 10.1 1.2 14.4 108.8 Table III. HF-LPME/LC-DAD inter-day precision, accuracy, recovery, enrichment factor and intra-day precision and accuracy Drugs Inter-day Intra-day Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 Extraction efficiency (%) n = 3 Enrichment factor Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 OME 0.2 14.3 91.5 3.47 1.49 0.3 12.1 99.9 1.0 8.0 100.1 17.7 7.56 0.9 12.2 93.5 2.0 13.0 109.4 19.2 8.24 1.2 7.8 106.2 PAN 0.2 5.2 106.2 8.01 3.43 0.3 13.8 101.5 1.0 13.0 101.0 25.5 10.9 0.9 4.1 106.1 2.0 12.3 102.4 25.6 10.9 1.2 13.5 116.1 LANSO 0.2 9.8 92.6 17.6 7.35 0.3 18.2 127.3 1.0 9.1 108.1 30.6 13.1 0.9 12.9 110.4 2.0 4.4 87.7 23.6 10.1 1.2 14.4 108.8 Drugs Inter-day Intra-day Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 Extraction efficiency (%) n = 3 Enrichment factor Spiked concentration (μg/mL) RSD (%) n = 3 Accuracy (%) n = 3 OME 0.2 14.3 91.5 3.47 1.49 0.3 12.1 99.9 1.0 8.0 100.1 17.7 7.56 0.9 12.2 93.5 2.0 13.0 109.4 19.2 8.24 1.2 7.8 106.2 PAN 0.2 5.2 106.2 8.01 3.43 0.3 13.8 101.5 1.0 13.0 101.0 25.5 10.9 0.9 4.1 106.1 2.0 12.3 102.4 25.6 10.9 1.2 13.5 116.1 LANSO 0.2 9.8 92.6 17.6 7.35 0.3 18.2 127.3 1.0 9.1 108.1 30.6 13.1 0.9 12.9 110.4 2.0 4.4 87.7 23.6 10.1 1.2 14.4 108.8 In the inter-day assay, the RSD (n = 3) values were <13.8% for all concentrations (0.3, 0.9 and 1.2 μg/mL) for OME and PAN, while for LANSO the RSD was 18.2%. However, as discussed above, LANSO is the analyte with the lowest stability in acid conditions and, as consequence, showed lower sensitivity in the calibration curve. Considering the measured accuracy, the inter-day experiments were between 93.5 and 106.2% for OME and PAN and from 108.8% to 127.3% for LANSO (Table III). The stability of the plasma samples spiked with drugs at different concentrations (0.2, 1.0 and 2.0 μg/mL) was monitored for 8 h (the time required for sample preparation and analysis in a single day) at ambient temperature, 25°C. No statistically significant differences were observed for the three different concentrations. The patient plasma samples were stored at −20°C for 2 weeks and these samples were stable for longer time periods under these conditions. The plasma samples spiked with PPI drugs at concentrations between 0.2 and 1.0 μg/mL were stable after three freeze-thaw cycles (in each freeze/thaw cycle, the samples were frozen at −20°C for 24 h, thawed and then maintained at ambient temperature for 1 h). The working solutions kept at −18°C and protected from light were stable for over 12 months. Method application In order to evaluate the proposed HF-LPME/LC-DAD method for clinical use, the described protocol was applied to the analysis of plasma samples from patients in therapy with the studied PPI drugs. The peak shapes and resolution were very similar to those obtained using spiked blank plasma and no interference was apparent. These plasma samples were collected from patients in therapy with OME (20 mg/day) and PAN sodium sesquihydrate (20 mg/day), respectively. The drug concentrations found in these samples were 1.12 and 1.15 μg/mL for OME (data not shown) and 1.92 μg/mL for PAN (Figure 5C). The plasma concentration values are within the linear range of the method, which was considered suitable based on a maximal plasma drug concentration between 0.2 and 2.0 μg/mL. Discussion LC-DAD conditions Several different elution conditions were evaluated for the simultaneous separation of the PPI drugs on the C18 column in order to obtain good resolution with a short analysis time. Initially, acetonitrile-water was used as the mobile phase, however, peak broadening was observed under these conditions. Thus, different mobile phase compositions incorporating phosphate (10 mol/L, pH at 3.0; 5.0; 6.0 and 7.0) and borate buffer solutions (10 mol/L, pH at 9.0) and acetonitrile were investigated, varying the acetonitrile ratio between 50 and 65% (v/v). Under alkaline conditions (pH 9.0) the analytes are predominantly in anionic form, while at pH 3.0 they are mostly cationic. Under these elution conditions, the retention times of these ionic forms of the drugs were close to each other and to the column dead volume, evidencing low selectivity. Due to the pKa values of the analytes, neutral forms are in the majority in the pH range 5.0–7.0. Thus, mobile phase compositions with acetonitrile and phosphate buffer solutions (10 mol/L) in this pH range were explored. Although asymmetric peaks were observed, improved resolution was achieved with phosphate buffer solution at pH 5.0 and acetonitrile (60:40 v/v) with a short 10-min chromatographic run. The addition of triethylamine (0.01%) in the mobile phase was tested, but no improvement in the peak symmetry was observed. As discussed by Santos Neto (34), peak distortions may occur when the solvent sample has a higher elution strength than the mobile phase. The PPI mix working solution was prepared in methanol, which has a higher elution strength than the combination of pH 5.0 phosphate buffer solution and acetonitrile (60:40 v/v). Hence, an improvement in the peak shape of the analytes was achieved by drying the methanol-PPI sample and redissolving it in the selected mobile phase. Based on literature, the wavelengths of 270, 285, 294 and 302 nm were investigated for evaluation of the maximum absorption of the analytes (2, 35, 36). It was confirmed that the λmax for OME was at 302 nm, while the maximum absorption for PAN and LANSO was at 285 nm (Figure 6). The IS λmax was at 270 nm, with significant absorption at 285 nm. Hence, for study of the HF-LPME parameters, 285 nm was selected for all drugs, although for validation and application in real samples the wavelength 302 nm was preferred for OME in order to improve the peak intensity (better detectability). Figure 6. View largeDownload slide Spectral UV of PPI drugs omeprazole, pantoprazole and lansoprazole with the wavelength selected. Figure 6. View largeDownload slide Spectral UV of PPI drugs omeprazole, pantoprazole and lansoprazole with the wavelength selected. Evaluation of the HF-LPME parameters Selection of the organic solvent is a crucial parameter as it is the extractor phase in both HF-LPME extraction modes. A suitable solvent should have low solubility in water to prevent leakage into the aqueous solution and maintain stability at the pores of the hydrophobic fiber. It is also advantageous to have low volatility in the three-phase mode, to avoid evaporation during extraction (24, 37). Five organic solvents that are commonly used in the two and three-phase methods were evaluated in triplicate: hexane, toluene, 1-octanol, dichloromethane and butyl acetate (25). Of these, butyl acetate and 1-octanol met the requirements for low volatility and low solubility in water for the three-phase mode process. When toluene, dichloromethane and hexane were used, solvent losses were observed during sample preparation and it was not possible to collect reproducible volumes of the acceptor phase. Between butyl acetate and 1-octanol, only 1-octanol satisfactorily extracted the PPI drugs and IS in three-phase mode in this study. Because an alkaline aqueous acceptor phase and an acidic donor phase were employed, butyl acetate hydrolysis may have occurred in one of these environments, as discussed in the literature (38, 39). Thus, 1-octanol was selected as the supported organic solvent. The pH of the donor phase (sample solution) was adjusted to be in the range 5.0–7.0 to maintain the analytes predominantly at neutral charge and improve the mass transfer into the organic solvent. Lower pH values cause the analytes to be in cationic form and as a consequence they may become degraded, since the PPI drugs are acid labeled. Therefore, the pH of the donor phase was adjusted with phosphate buffer solutions (10 mol/L) of pH 5.0, 6.0 and 7.0. The highest extraction recoveries were obtained with the donor phase at pH 5.0 or 6.0, (the difference between them not being significant) (Figure 3A); while a decrease in recovery was detected at pH 7.0. This might be attributed to deprotonation occurring at higher pH, causing ionization of the drugs. As the mobile phase of the chromatographic method was composed by phosphate buffer at pH 5.0 and acetonitrile, pH 5.0 phosphate buffer solution was selected for the donor phase to facilitate injection of the sample into the analytical system. The pH of the aqueous acceptor phase used in three-phase mode is modified to ensure that the analyte molecules are predominantly in the anionic form, and thus prevent them from re-entering the organic phase (24). Borate buffer solutions (10 mol/L) at pH 9.0 and 10.0 were evaluated for this purpose. With a pH 9.0 acceptor phase, a lower extraction recovery was observed for all drugs, compared with pH 10.0 (Figure 3A). This behavior might be attributed to the fact that the ionized forms of the PPI drugs are more favored when the pH is increased. Accordingly, the selected acceptor phase was borate solution (10 mol/L) at pH 10. Using the selected organic solvent and the optimum pH conditions for the donor and acceptor phases, the influence of the extraction time was evaluated in the range of 10–45 min. Distinct behaviors were observed for the different analytes, which may be associated with their physical and chemical differences based on the nature of the substituents on the PPI drug molecules (7). As a result (Figure 3B), it was observed that PAN reached partition equilibrium after 30 min of extraction. OME also reached mass transfer equilibrium at this extraction time, however, there was a decrease in the peak area beginning at 15 min. Finally, LANSO did not reach a plateau in the tested time period and its RSD values increased from 17% at 10 min to 54% at 45 min. Considering these results, an extraction time of 15 min was selected. These results show that it is possible to employ the HF-LPME technique under non-equilibrium conditions for PPI drug extraction in the three-phase mode, despite their instability under acid conditions. Magnetic stirring improves the mass transfer of analytes from the plasma sample to the hollow-fiber. It is known that for solid-phase microextraction, the effect of the static layer zone close to the fiber is reduced by stirring (40). Similarly, for the hollow-fiber in the present study, the samples were stirred during extraction at a maximum rate of 750 rpm, without loss of extraction solvent from the fiber. The possibility of salting out effect was also investigated by increasing the NaCl concentration between 0 and 30% (w/v) in the HF-LPME extraction of the PPI drugs. As shown in Figure 3C, there was a rise in the extraction efficiency with increasing salt concentration up to 5% and a reduction >10%. The addition of salt may decrease analyte solubility since the water molecules of the sample hydrate ionic salt molecules and the reduction in extraction efficiency is explained by the fact that electrostatic interactions can occur between polar molecules and salt ions (41). In addition, increased salt concentration may increase the donor-phase viscosity and lead to emulsion formation. Based on this, a salt concentration of 5% (w/v) was selected. Analytical validation As shown in Figure 3D, human plasma samples showed decreased extraction efficiency, indicating an effect of matrix compounds, mainly plasma proteins. As a result, the application of three sample treatments was evaluated: protein filtration, protein precipitation and addition of the organic modifier methanol. Methanol was tested as when lower recoveries are observed in plasma than in water, the main interactions might be hydrophobic and the addition of an organic modifier such as methanol can suppress this binding (33). Precipitation or filtration did not improve the extraction efficiency. This may be because of drug–protein binding the analytes may be precipitated and filtered out along with the proteins. On the other hand, the addition of methanol showed an improvement in extraction recovery at a human plasma:methanol ratio of 1:1 (v/v) and decreases with higher proportions of methanol. The addition of methanol may affect the distribution of the analyte in the aqueous and organic phases, decreasing the extraction recovery (42). Although the addition of methanol to plasma did not improve the extraction recovery to a level comparable with that in ultrapure water or synthetic plasma, a significant increase in recovery was achieved compared with plasma samples without treatment, as shown in Figure 4. Since the RSD at the lowest concentration level (0.2 μg/mL) was <20% and showed accuracy in the range of 80–120%, this could be considered as the lower LOQ according to US-FDA recommendations. The LOQ is higher than in literature references concerning PPI drug determination, which range from 2.5 to 20 ng/mL. However, as previously mentioned, these drugs are typically extracted using conventional exhaustive techniques such as LLE and SPE (17, 43, 44) and, in some cases, with calibration curves measured in solvent or in the mobile phase. Also, highly sensitive analytical techniques such as LC-MS and LC-MS/MS are commonly used (16, 45). Beyond that, it should be noted that there are no documented studies employing HF-LPME for PPI drug determination in human plasma in a simple analytical system such as LC-DAD. Furthermore, the maximal plasma concentrations of the target drugs range between 0.5 and 1.5 μg/mL in the interval of 1–4 h (46, 47). Based on this information, an analytical method should be able to determinate these drugs in the concentration range 0.2–2.0 μg/mL assuming sampling at the time of maximal plasma concentration, the LOQ obtained in this paper is in this concentration range. Extraction efficiency values were satisfactory, in the range of 3.5–30%, as was the enrichment factor (between 1.5 and 13), considering the significant matrix effect. It can also be highlighted that HF-LPME is not an exhaustive but an equilibrium technique (42), however, the extraction should be performed rapidly as a consequence of the acid instability of the analytes. Recoveries with SPE or LLE techniques of these drugs are between 50 and 110% (17, 18, 45, 47). The developed method and sample preparation can be performed under non-equilibrium conditions with a mere 15 min of extraction time, while achieving the proper accuracy and precision. Furthermore, studies employing HF-LPME typically report recoveries near to 10% for biological samples (48). Moreover, there are also reports in the literature with recoveries <10%, for example, absolute recoveries in the range of 4.4–8.9% for the determination of cannabinoids in human hair by GC-MS/MS (49). Method application This application of the method shows that is possible to employ the HF-LPME microextraction technique for PPI drug determination in plasma samples, contributing to studies of the adverse effects of these drugs in long-term therapy. Conclusions In this study, an HF-LPME/LC-DAD method was developed, validated and applied for the determination of OME, PAN and LANSO in human plasma samples. This allowed a low cost, rapid, sensitive, selective and reproducible methodology for the determination of these drugs. In addition, the requirement for organic solvents is reduced and the sample volume used is lower than in conventional techniques, which is important for invasive samples such as human plasma. Although the matrix complexity, it was possible to apply the HF-LPME technique with reduced matrix effects, in fast analyses, and obtain clean extracts without interferents, because the pore size of the fiber (0.2-μm) can block the passage of larger molecules such as plasma proteins and decrease the interference of macromolecules in chromatographic analysis. The sample preparation could also be performed under non-equilibrium conditions with suitable accuracy and precision. The investigation of the HF-LPME parameters was crucial to allow the determination of PPI drugs in acid conditions. This study contributes to knowledge about the determination of these types of drugs using miniaturized techniques and will enable studies concerning adverse drug effects. Finally, the HF-LPME/LC-DAD method was successfully applied in human plasma samples from patients undergoing therapy with OME and PAN. 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Published: Mar 28, 2018

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