Advance Access Publication Date: 28 March 2018Article
Article
Evaluation of a Hollow-Fiber Liquid-Phase
Microextraction Technique for the Simultaneous
Determination of PPI Drugs in Human Plasma by
LC-DAD
Rayta Paim Horta, Bianca do Amaral, Patricio Guillermo Peralta-Zamora,
and Bruno José Gonçalves Silva*
Departamento de Química, Universidade Federal do Paraná, C.P. 19081, Curitiba 81531-980, Brazil
*Author to whom correspondence should be addressed. Email: [email protected]
Received 28 July 2017; Revised 1 December 2017; Editorial Decision 4 March 2018
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 15min; 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 concentra-
tion 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 pa-
tients undergoing therapy with the PPI drugs.
Introduction
Proton pump inhibitors (PPI) are some of the most widely prescribeddrugs used in the treatment of disorders related to gastric acid secretion(1). PPIs, which include omeprazole (OME), pantoprazole (PAN) andlansoprazole (LANSO), are weak basic prodrugs that are converted totheir active form in acid conditions. These activated forms block thegastric H+/K+ATPase (proton pump enzyme) which is responsible forthe final step of the acid secretion process (2–4). As they have pKa1 andpKa2 values around 4 and 9, respectively (1), their activation can occurin the acid conditions of the stomach parietal cells (pH 1.3) that is theirsite of action (5). This is reflected in the fact that this class of drugs ismore effective than others in the suppression of acid secretion (6).
OME, PAN and LANSO have the same general mechanism ofaction and are unstable in acid environments but their different sub-stituents on the pyridine and benzimidazole groups (Figure 1) resultin some distinct properties (7). Concerning their acid stability, forexample, the degradation rate is different and the stability order is:PAN > OME > LANSO (3). Another pharmacokinetic difference isthe time taken to reach peak plasma concentration that is in therange of 0.5–3.5 h for OME, 1.7 h for LANSO and 1.1–3.1 h forPAN (8). All PPIs are rapidly absorbed and are highly plasma pro-tein bound, which results in a low volume of distribution (9). Theproportion of the drug bound to protein is 95% for OME, 98% forLANSO and 97% for PAN (8, 10, 11).
Recent studies have made an association between chronic PPItherapy and potential adverse effects such as dementia, an increasedrisk of bone fractures and deficiency of vitamin B12 among others(12–15). For this reason, it is important to develop new methodolo-gies to determine these drugs in biological matrices in order to evalu-ate their effects. In the literature, there are few works where thesimultaneous determination of PPI drugs in human plasma has beenperformed, as reported by Bharathi and coworkers (2). Most studiesconcern one PPI drug and its enantiomers with their respective meta-bolites. 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 tandemmass spectrometry (LC-MS/MS) or ultraviolet detection (LC-UV orLC-DAD). However, these conventional extraction methods havesome disadvantages, such as the consumption of large volumes oftoxic organic solvents, the requirement of costly cartridges and theyare time-consuming (19–21).
The hollow-fiber liquid-phase microextraction (HF-LPME) tech-nique has received some attention since it uses low volumes of organicsolvents to extract organic compounds from small amounts of aqueoussamples (22). Furthermore, the technique employs a porous hydro-phobic hollow fiber. This inexpensive disposable device eliminates thecarry-over effect and its pore size can block the passage of larger mo-lecules such as plasma proteins (23, 24).
HF-LPME was introduced in 1999 (24) and performs the extrac-tion of compounds from a donor phase (sample) through an organicsolvent (extractor phase) into an acceptor phase inside the lumen ofa hollow fiber. This acceptor phase can be the same extractororganic solvent as that immobilized in the fiber pores, correspondingto the two-phase mode; alternatively, it can be an aqueous solutionwhen the technique is used in the three-phase mode (20). Becausethe extractor and acceptor phases are, respectively, immobilized inthe 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 fordrug 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 andLANSO in human plasma samples.
Differently to previous studies concerning the determination of PPIdrugs, in this paper we developed a miniaturized method (HF-LPME)for the simultaneous determination of three PPI drugs in humanplasma by LC-DAD: OME, PAN and LANSO. The chromatographicand extraction parameters were evaluated in order to enable theextraction of these acid labeled and highly protein bound drugs fromthis complex matrix. Moreover, the method was validated and appliedto 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 per-formance liquid chromatography (HPLC) grade acetonitrile andmethanol were obtained from J.T. Baker® (Phillipsburg, USA).Dibasic potassium phosphate (Vetec®, Rio de Janeiro, Brazil) andboric acid (Isofar®, Rio de Janeiro, Brazil) were used to preparebuffer solutions in ultrapure water purified by a Milli-Q Millipore®
system (18MΩ at 25°C) (São Paulo, Brazil). Working standard drugsolutions were prepared by the diluting 1mg/mL methanol stock so-lutions to the appropriate volume with methanol and kept at −18°Cprotected from light. Hexane, toluene and 1-octanol were purchasedfrom Sigma-Aldrich® (São Paulo, Brazil). Butyl acetate (F. Maia®,
Figure 1. Chemical structure of selected PPI drugs.
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São Paulo, Brazil) and dichloromethane (Vetec®, Rio de Janeiro, Brazil)were also evaluated as organic solvents. pH adjustments were madeusing diluted solutions of sodium hydroxide (Proquímios®, Rio deJaneiro, 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® 1260Infinity HPLC system with quaternary pump and diode array detection.The separation was achieved using an Agilent® Microsorb MV100-5C18 column (250mm × 4.6mm, 5 μm), preceded by an Agilent® guardC18 column, at 30°C. The mobile phase consisted of phosphate buffersolution (10mol/L, pH 5.0): acetonitrile 60:40 (v/v) in isocratic mode,at a flow-rate of 1.0mL/min, with only 10min of chromatographicanalyses. The mobile phase was filtered and degassed prior to use andthe wavelengths used for detection were 285 and 302nm.
Plasma samples
Evaluation of the HF-LPME parameters was performed using syn-thetic plasma consisted by the chlorides of sodium (145mmol/L),potassium (4.5mmol/L), calcium (32.5mmol/L) and magnesium (0.8mmol/L) in addition to urea (2.5mmol/L) and glucose (4.7mmol/L),as described in the literature (29). The synthetic plasma solution wasstored at −20°C for up to 2 weeks. Plasma from healthy volunteersthat had not been subjected to any pharmacological treatment for atleast 72 h (blank plasma) was supplied by the Clinical AnalysisLaboratory Unit of the Clinical Hospital at the Federal University ofParaná, Brazil. The plasma samples were stored at −20°C for up to 3months and were used for validation of the method. The HF-LPME/LC-DAD method was applied to human plasma samples from six pa-tients in PPI therapy. Written consent was obtained prior to the study.Blood samples were taken until 6 h after the final administration ofthe drug. It should be emphasized that the study did not interfere withthe clinical conduct adopted for the patients. The study was approvedby Human Research Ethics Committee of the HC at the FederalUniversity of Paraná (Curitiba, Brazil).
Methods
HF-LPME apparatus and procedure
All HF-LPME experiments were performed using Q3/2 Accurelpolypropylene hollow fiber (600 μm i.d., 200 μm wall thickness and0.2 μm pore size) purchased from Membrana® (Wuppertal, Germany).Before use, the hollow fiber was cleaned with acetone in an ultrasonicbath for 5min. After drying, the fiber was cut to a length of 8 cm andthen soaked in the organic solvent for 10 s to impregnate the pores.Excess solvent was removed by washing in water for 20 s in anultrasonic bath. Subsequently, the fiber was formed into a U-shapeand connected to two 25 μL liquid chromatographic microsyringes(model 702SNR—Hamilton® Reno, NV) and 25 μL of acceptorphase was injected into the lumen. Before each extraction, the mi-crosyringes were washed 10 times with acetone and the acceptorphase, in sequence. The fiber was then immersed into the sample(donor phase) and the extraction was performed with magnetic stir-ring at 750 rpm (Biomixer® model 78HW-1) for 15min at roomtemperature (Figure 2).
For parameters experiments, the donor phase consisted of equal vo-lumes of synthetic plasma and different buffer solutions. In a glass vial(10mL, Shimadzu®, Japan), 100 μL of IS (20 μg/mL) and 200 μL ofdrug standard mix solution (100 μg/mL) were mixed, and the methanol
was evaporated to dryness. The standards were then reconstituted in5mL of buffer solution and added to 5mL of the synthetic plasma, re-sulting in a final concentration level of 2.0 and 0.2 μg/mL for the PPIdrugs and IS, respectively. For analytical validation and application inreal samples, the donor phase was composed by 1.0mL of humanplasma and methanol with the addition of phosphate buffer (10mol/L,pH 5) containing 5% NaCl (w/v), to a final volume of 4.5mL. Afterextraction, the microsyringes and the fiber were taken out of the sam-ple and one of the syringes was used to collect the acceptor phase fromthe lumen of the fiber. The acceptor phase collected in parameters eval-uation was 10 μL, which was made up to 200 μL with mobile phaseand vortexed. In order to increase the enrichment factor, in validationand application experiments, the acceptor phase volume was 15 μL,with a final volume of 100 μL (with mobile phase). Finally, 50 μL ofthis extract was injected into the LC-DAD system.
HF-LPME parameters
The investigation of HF-LPME conditions was performed accordingto the HF-LPME procedure described. Triplicate or quadruplicateanalyses were performed for all experiments and the mean valueswere used to plot the results. The choice of the hollow-fiber devicewas the first step. A low cost porous hydrophobic hollow-fiber wasselected because it allows the separation of the donor and acceptorphases during the extraction procedure. In addition, as reported pre-viously, the pore size of the fibers (0.2 μm) can block the passage oflarger molecules such as plasma proteins (23, 24), decreasing inter-ference in the chromatographic analysis caused by macromolecules.
The influence of the matrix pH on the PPI extraction was investi-gated. For this purpose, several pH values were investigated: 5.0,6.0 and 7.0 (phosphate buffer 10mmol/L) for the donor phase; andpH 9.0 and 10.0 (borate buffer 10mol/L) for the acceptor phase.
The effects of the ionic strength of the matrix solution (addition of0; 5, 10, 20 and 30% of NaCl, w/v) and the equilibrium time (10, 15,30 and 45min) on the PPI extraction efficiency were also investigatedat room temperature. Different extraction solvents (1-octanol, butylacetate, toluene, hexane and dichloromethane) were also evaluated forimpregnation of the porous fibers.
Analytical validation
Validation of the HF-LPME/LC-DAD method was carried out underthe selected conditions. Blank plasma samples spiked with the IS
Figure 2. Schematic illustration of the HF-LPME apparatus for extraction.
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and analytes at various concentrations were used. The concentrationrange included the therapeutic range of the PPI drugs. Firstly, it wasnecessary to reduce the effect of the matrix since there was a signifi-cant binding of proteins (e.g., albumin) to the analytes. Thus, filtra-tion in a 13-mm polycarbonate syringe filter holder (cellulose nitratemembrane 0.45 μm pores—Sartorius Stedium® Goettingen,Germany) and protein precipitation were evaluated, based on pub-lished methods in the literature (30, 31), and the solvent was chan-ged from acetonitrile to methanol. Also, the addition of the organicmodifier methanol to the donor phase to suppress protein bindingwithout precipitation was evaluated.
The selectivity of the method against endogenous interferencewas verified by examining the chromatograms obtained after the mi-croextraction of blank plasma samples from at least 10 differentsources. Furthermore, the selectivity of the method was investigatedby comparison of the retention times among the analytes and othersubstances. For this purpose, the possibility of co-elution of 11potential interfering compounds: caffeine, zidovudine, hydrochloro-thiazide, diazepam, chloramphenicol, carbamazepine, acetylsalicylicacid, paracetamol, amoxicillin, diclofenac and ibuprofen was evalu-ated. The acceptance criterion for this study was based on theabsence of substances with interfering peaks at the retention timesof the drugs of interest.
The linearity and limit of quantification (LOQ) were evaluatedusing calibration curves constructed by analyzing spiked humanplasma samples after extraction by HF-LPME. This study was evalu-ated 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 wasmaintained at 0.20 μg/mL. The LOQ was determined based on US-FDA recommendations (32).
Accuracy, intra- and inter-day precision studies were performedby analyzing human plasma samples after HF-LPME/LC-DAD, intriplicate, with three different concentrations (low-, medium- andhigh-level) of PPI. For accuracy and intra-day precision, the analyteconcentration 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 ofHo and coworkers (33). The enrichment factor was calculated followingthe methods in a previous study by Rasmussen and Perdersen-Bjergaard(25).
In the study of stability, the bench stability of the analytes in thematrix at ambient temperature, the storage (freezer) stability of theplasma sample, as well as the stability of the pure standard solutionsin storage and after freeze-thaw were evaluated.
Results
LC-DAD conditions and HF-LPME parameters
The selected chromatographic conditions were a mobile phase con-sisting of phosphate buffer solution (10mol/L, pH 5.0): acetonitrile60:40 (v/v) in isocratic mode. The flow-rate was 1.0mL/min at atemperature of 30°C, with detection at λ = 285 and 302 nm. Therobustness of the method was evaluated about column temperature
Figure 3. 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.
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control (without and 30°C) and by mix standard PPI drugs solutionsinjections along 6 months and no significant changes were observed.
The investigation of the HF-LPME parameters is shown in Figure 3and the conditions selected were 1-octanol as solvent, phosphate buffer atpH 5 (10mmol/L) as donor phase, borate buffer at pH 10 (10mmol/L)as acceptor phase, extraction time of 15min, stirring rate of 750 rpm andNaCl at 5% (w/v). Using the selected conditions, analytical validation ofthe HF-LPME/LC-DAD method was performed.
Analytical validation
First, it was necessary to study the matrix effect since protein bindingmay result in low recovery from human plasma samples. This effectwas evaluated by comparing extraction recoveries in ultrapure water,synthetic plasma and human plasma samples (Figure 3D). Because ofthe plasma proteins, the application of three sample treatments wasevaluated: protein filtration, protein precipitation and addition meth-anol and being that the latter was selected (Figure 4).
The developed HF-LPME/LC-DAD method showed high selectivity.The retention times of the drugs of interest were different compared withthose of possible interfering compounds analyzed under the same chro-matography conditions in concentrations around 1 μg/mL (Table I).Furthermore, the selectivity of the method is also demonstrated by repre-sentative chromatograms of drug-free plasma after HF-LPME (blanksample) 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 arefree from interfering peaks due to endogenous compounds co-elutingwith the drugs of interest. This result demonstrates the ability of the fiberto block the access of the plasma protein to the acceptor phase, as previ-ously indicated.
The linearity of the HF-LPME method was determined usingdrug-free plasma spiked with the PPI drugs in the range from 0.2 to2.0 μg/mL. The analyses after HF-LPME were performed in quadru-plicate. This interval was linear, with correlation coefficients >0.991
and relative standard deviations (RSD) below 15% for all concen-trations in all cases (Table II). These results show that the developedmethod allows the determination of PPI in plasma over a wide rangeof concentrations.
Moreover, the method exhibited suitable accuracy and precision.The RSD of intra-day experiments (n = 3) was <14.3% for all analytesat all three concentrations evaluated (0.2, 1.0 and 2.0 μg/mL). Theaccuracy at these concentrations was in the range of 87.7–108.1% andrecovery values were in the range of 3.47–30.6% (Table III).
In the inter-day assay, the RSD (n = 3) values were <13.8% forall 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
Figure 4. 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).
Table I. Retention time of analytes, IS and possible interfering
Compounds in bold are the analytes studied in this work.ND: not detected until 20 min of chromatographic analysis.
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above, LANSO is the analyte with the lowest stability in acid condi-tions and, as consequence, showed lower sensitivity in the calibrationcurve. Considering the measured accuracy, the inter-day experimentswere between 93.5 and 106.2% for OME and PAN and from108.8% to 127.3% for LANSO (Table III).
The stability of the plasma samples spiked with drugs at differentconcentrations (0.2, 1.0 and 2.0 μg/mL) was monitored for 8 h (thetime required for sample preparation and analysis in a single day) atambient temperature, 25°C. No statistically significant differenceswere observed for the three different concentrations. The patientplasma samples were stored at −20°C for 2 weeks and these sampleswere stable for longer time periods under these conditions. Theplasma samples spiked with PPI drugs at concentrations between0.2 and 1.0 μg/mL were stable after three freeze-thaw cycles (in eachfreeze/thaw cycle, the samples were frozen at −20°C for 24 h, thawedand then maintained at ambient temperature for 1 h). The working so-lutions kept at −18°C and protected from light were stable for over12 months.
Method application
In order to evaluate the proposed HF-LPME/LC-DAD method forclinical use, the described protocol was applied to the analysis ofplasma samples from patients in therapy with the studied PPI drugs.The peak shapes and resolution were very similar to those obtainedusing spiked blank plasma and no interference was apparent. Theseplasma samples were collected from patients in therapy with OME(20 mg/day) and PAN sodium sesquihydrate (20 mg/day), respec-tively. The drug concentrations found in these samples were 1.12and 1.15 μg/mL for OME (data not shown) and 1.92 μg/mL forPAN (Figure 5C). The plasma concentration values are withinthe linear range of the method, which was considered suitablebased on a maximal plasma drug concentration between 0.2 and2.0 μg/mL.
Discussion
LC-DAD conditions
Several different elution conditions were evaluated for the simultaneousseparation of the PPI drugs on the C18 column in order to obtain goodresolution with a short analysis time. Initially, acetonitrile-water wasused as the mobile phase, however, peak broadening was observedunder these conditions. Thus, different mobile phase compositionsincorporating phosphate (10mol/L, pH at 3.0; 5.0; 6.0 and 7.0) andborate buffer solutions (10mol/L, pH at 9.0) and acetonitrile wereinvestigated, varying the acetonitrile ratio between 50 and 65% (v/v).Under alkaline conditions (pH 9.0) the analytes are predominantly inanionic form, while at pH 3.0 they are mostly cationic. Under theseelution conditions, the retention times of these ionic forms of the drugs
Figure 5. 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.
Table II. HF-LPME/LC-DAD calibration parameters
Drugs Linear range Determination coefficient (R2) Intercept Slope RSD% LOQ (0.2 μg/mL) n = 3
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were close to each other and to the column dead volume, evidencinglow selectivity.
Due to the pKa values of the analytes, neutral forms are in themajority in the pH range 5.0–7.0. Thus, mobile phase compositionswith acetonitrile and phosphate buffer solutions (10mol/L) in this pHrange were explored. Although asymmetric peaks were observed,improved resolution was achieved with phosphate buffer solution at pH5.0 and acetonitrile (60:40 v/v) with a short 10-min chromatographicrun. The addition of triethylamine (0.01%) in the mobile phase wastested, but no improvement in the peak symmetry was observed. As dis-cussed by Santos Neto (34), peak distortions may occur when the sol-vent sample has a higher elution strength than the mobile phase. ThePPI mix working solution was prepared in methanol, which has a high-er elution strength than the combination of pH 5.0 phosphate buffersolution and acetonitrile (60:40 v/v). Hence, an improvement in thepeak shape of the analytes was achieved by drying the methanol-PPIsample and redissolving it in the selected mobile phase.
Based on literature, the wavelengths of 270, 285, 294 and302 nm were investigated for evaluation of the maximum absorp-tion of the analytes (2, 35, 36). It was confirmed that the λmax forOME was at 302 nm, while the maximum absorption for PAN andLANSO was at 285 nm (Figure 6). The IS λmax was at 270 nm, withsignificant absorption at 285 nm. Hence, for study of the HF-LPMEparameters, 285 nm was selected for all drugs, although for valida-tion and application in real samples the wavelength 302 nm was pre-ferred for OME in order to improve the peak intensity (betterdetectability).
Evaluation of the HF-LPME parameters
Selection of the organic solvent is a crucial parameter as it is theextractor phase in both HF-LPME extraction modes. A suitable sol-vent should have low solubility in water to prevent leakage into theaqueous solution and maintain stability at the pores of the hydro-phobic fiber. It is also advantageous to have low volatility in thethree-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 inwater for the three-phase mode process. When toluene, dichloro-methane and hexane were used, solvent losses were observed duringsample preparation and it was not possible to collect reproducible vo-lumes of the acceptor phase. Between butyl acetate and 1-octanol, only
1-octanol satisfactorily extracted the PPI drugs and IS in three-phasemode in this study. Because an alkaline aqueous acceptor phase and anacidic donor phase were employed, butyl acetate hydrolysis may haveoccurred 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 bein the range 5.0–7.0 to maintain the analytes predominantly at neu-tral charge and improve the mass transfer into the organic solvent.Lower pH values cause the analytes to be in cationic form and as aconsequence they may become degraded, since the PPI drugs areacid labeled. Therefore, the pH of the donor phase was adjustedwith phosphate buffer solutions (10mol/L) of pH 5.0, 6.0 and 7.0.The highest extraction recoveries were obtained with the donorphase at pH 5.0 or 6.0, (the difference between them not being sig-nificant) (Figure 3A); while a decrease in recovery was detected atpH 7.0. This might be attributed to deprotonation occurring athigher pH, causing ionization of the drugs. As the mobile phase ofthe chromatographic method was composed by phosphate buffer atpH 5.0 and acetonitrile, pH 5.0 phosphate buffer solution wasselected for the donor phase to facilitate injection of the sample intothe analytical system.
The pH of the aqueous acceptor phase used in three-phase modeis modified to ensure that the analyte molecules are predominantlyin the anionic form, and thus prevent them from re-entering theorganic phase (24). Borate buffer solutions (10mol/L) at pH 9.0 and10.0 were evaluated for this purpose. With a pH 9.0 acceptor phase,a lower extraction recovery was observed for all drugs, comparedwith pH 10.0 (Figure 3A). This behavior might be attributed to thefact that the ionized forms of the PPI drugs are more favored whenthe pH is increased. Accordingly, the selected acceptor phase wasborate solution (10mol/L) at pH 10.
Using the selected organic solvent and the optimum pH condi-tions for the donor and acceptor phases, the influence of the extrac-tion time was evaluated in the range of 10–45min. Distinctbehaviors were observed for the different analytes, which may beassociated with their physical and chemical differences based on thenature of the substituents on the PPI drug molecules (7). As a result(Figure 3B), it was observed that PAN reached partition equilibriumafter 30min of extraction. OME also reached mass transfer equilib-rium at this extraction time, however, there was a decrease in thepeak area beginning at 15min. Finally, LANSO did not reach a pla-teau in the tested time period and its RSD values increased from17% at 10min to 54% at 45min. Considering these results, anextraction time of 15min was selected. These results show that it is
Table III. HF-LPME/LC-DAD inter-day precision, accuracy, recovery, enrichment factor and intra-day precision and accuracy
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possible to employ the HF-LPME technique under non-equilibriumconditions for PPI drug extraction in the three-phase mode, despitetheir instability under acid conditions.
Magnetic stirring improves the mass transfer of analytes from theplasma sample to the hollow-fiber. It is known that for solid-phasemicroextraction, the effect of the static layer zone close to the fiber isreduced by stirring (40). Similarly, for the hollow-fiber in the presentstudy, the samples were stirred during extraction at a maximum rateof 750 rpm, without loss of extraction solvent from the fiber.
The possibility of salting out effect was also investigated byincreasing the NaCl concentration between 0 and 30% (w/v) in theHF-LPME extraction of the PPI drugs. As shown in Figure 3C, therewas a rise in the extraction efficiency with increasing salt concentra-tion up to 5% and a reduction >10%. The addition of salt may
decrease analyte solubility since the water molecules of the samplehydrate ionic salt molecules and the reduction in extraction effi-ciency is explained by the fact that electrostatic interactions canoccur between polar molecules and salt ions (41). In addition,increased salt concentration may increase the donor-phase viscosityand lead to emulsion formation. Based on this, a salt concentrationof 5% (w/v) was selected.
Analytical validation
As shown in Figure 3D, human plasma samples showed decreasedextraction efficiency, indicating an effect of matrix compounds,mainly plasma proteins. As a result, the application of three sampletreatments was evaluated: protein filtration, protein precipitationand addition of the organic modifier methanol. Methanol was testedas when lower recoveries are observed in plasma than in water, themain interactions might be hydrophobic and the addition of anorganic 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 beprecipitated and filtered out along with the proteins. On the otherhand, the addition of methanol showed an improvement in extractionrecovery at a human plasma:methanol ratio of 1:1 (v/v) and decreaseswith higher proportions of methanol. The addition of methanol mayaffect the distribution of the analyte in the aqueous and organic phases,decreasing the extraction recovery (42). Although the addition of meth-anol to plasma did not improve the extraction recovery to a level com-parable with that in ultrapure water or synthetic plasma, a significantincrease in recovery was achieved compared with plasma samples with-out 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 beconsidered as the lower LOQ according to US-FDA recommenda-tions. The LOQ is higher than in literature references concerning PPIdrug determination, which range from 2.5 to 20 ng/mL. However, aspreviously mentioned, these drugs are typically extracted using con-ventional exhaustive techniques such as LLE and SPE (17, 43, 44)and, in some cases, with calibration curves measured in solvent or inthe mobile phase. Also, highly sensitive analytical techniques such asLC-MS and LC-MS/MS are commonly used (16, 45). Beyond that, itshould be noted that there are no documented studies employing HF-LPME for PPI drug determination in human plasma in a simple ana-lytical system such as LC-DAD. Furthermore, the maximal plasmaconcentrations of the target drugs range between 0.5 and 1.5 μg/mL inthe interval of 1–4 h (46, 47). Based on this information, an analyticalmethod should be able to determinate these drugs in the concentrationrange 0.2–2.0 μg/mL assuming sampling at the time of maximalplasma concentration, the LOQ obtained in this paper is in this con-centration range.
Extraction efficiency values were satisfactory, in the range of3.5–30%, as was the enrichment factor (between 1.5 and 13), con-sidering the significant matrix effect. It can also be highlighted thatHF-LPME is not an exhaustive but an equilibrium technique (42),however, the extraction should be performed rapidly as a conse-quence of the acid instability of the analytes. Recoveries with SPE orLLE techniques of these drugs are between 50 and 110% (17, 18,45, 47). The developed method and sample preparation can be per-formed under non-equilibrium conditions with a mere 15min ofextraction time, while achieving the proper accuracy and precision.Furthermore, studies employing HF-LPME typically report recover-ies near to 10% for biological samples (48). Moreover, there are
Figure 6. Spectral UV of PPI drugs omeprazole, pantoprazole and lansopra-
zole with the wavelength selected.
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also reports in the literature with recoveries <10%, for example,absolute recoveries in the range of 4.4–8.9% for the determinationof cannabinoids in human hair by GC-MS/MS (49).
Method application
This application of the method shows that is possible to employ theHF-LPME microextraction technique for PPI drug determination inplasma samples, contributing to studies of the adverse effects ofthese drugs in long-term therapy.
Conclusions
In this study, an HF-LPME/LC-DAD method was developed, valid-ated and applied for the determination of OME, PAN and LANSOin human plasma samples. This allowed a low cost, rapid, sensitive,selective and reproducible methodology for the determination ofthese drugs. In addition, the requirement for organic solvents isreduced and the sample volume used is lower than in conventionaltechniques, which is important for invasive samples such as humanplasma. Although the matrix complexity, it was possible to applythe HF-LPME technique with reduced matrix effects, in fast analy-ses, and obtain clean extracts without interferents, because the poresize of the fiber (0.2-μm) can block the passage of larger moleculessuch as plasma proteins and decrease the interference of macromole-cules in chromatographic analysis. The sample preparation couldalso be performed under non-equilibrium conditions with suitableaccuracy and precision. The investigation of the HF-LPME para-meters was crucial to allow the determination of PPI drugs in acidconditions. This study contributes to knowledge about the determi-nation of these types of drugs using miniaturized techniques and willenable studies concerning adverse drug effects. Finally, the HF-LPME/LC-DAD method was successfully applied in human plasmasamples from patients undergoing therapy with OME and PAN.
Funding
This work was supported by grants from Conselho Nacional de DesenvolvimentoCientífico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento dePessoal de Nível Superior (CAPES).
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