New flow injection method for quality control of dietary supplements containing l -carnitine using extraction mediated by sodium taurodeoxycholate coacervate coupled to molecular fluorescence

    New flow injection method for quality control of dietary supplements containing L-carnitine using extraction mediated by Sodium taurodeoxycholate coacervate coupled to molecular fluorescence Andrea C. Isaguirre, Gimena Acosta, Soledad Cerutti, Liliana P. Fernandez PII: DOI: Reference:

S0026-265X(16)30112-6 doi: 10.1016/j.microc.2016.06.025 MICROC 2515

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

29 March 2016 21 June 2016 24 June 2016

Please cite this article as: Andrea C. Isaguirre, Gimena Acosta, Soledad Cerutti, Liliana P. Fernandez, New flow injection method for quality control of dietary supplements containing L-carnitine using extraction mediated by Sodium taurodeoxycholate coacervate coupled to molecular fluorescence, Microchemical Journal (2016), doi: 10.1016/j.microc.2016.06.025

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ACCEPTED MANUSCRIPT New flow injection method for quality control of dietary supplements containing L-carnitine using extraction mediated by Sodium

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taurodeoxycholate coacervate coupled to molecular fluorescence

Instituto de Química de San Luis (INQUISAL), Centro Científico Tecnológico (CCT) San

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a

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Andrea C. Isaguirrea,b, Gimena Acostaa,c, Soledad Ceruttia,d, Liliana P. Fernandeza,d,*

Luis. UNSL-CONICET. Chacabuco 917, San Luis. ARGENTINA (5700) Área de Biología, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis.

Control de Calidad de Medicamentos, Área de Gestión en Calidad y Salud, Facultad de

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c

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Química, Bioquímica y Farmacia, Universidad Nacional de San Luis. d

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Área de Química Analítica, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis.

Author to whom correspondence should be addressed

e-mail: [email protected] (L.P. Fernández) 1

ACCEPTED MANUSCRIPT Abstract Carnitine (CAR) is an essential component of the tissue of animals, higher plants and many

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microorganisms; it plays an important role during the metabolism of the human body. CAR

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transfers fatty acids to the place of burning-mitochondria and aids the transformation of fats

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into energy and this way support overweight reduction and immediate physical performance, increases resistance from physical load and protect heart from overload. In

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this study, a new methodology for the determination of L- CAR by molecular fluorescence

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is described, after derivatization with a fluorescence active substance, 9-fluorenylmethyl chloroformate. An on line coacervation process was used in order to isolate the analyte of

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the complex matrix. The developed methodology was successfully applied to the control of

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carnitine content in dietary supplements. The detection limit and quantification obtained were 2.4x10-8 mol L-1 and 7.30x10-8 mol L-1; respectively. Recoveries between 82 and

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103% were obtained. At optimized working conditions, the sample throughput was 30 samples h-1 (without consider the required time for L-CAR derivatization). Thus short analysis and very low running costs comparing with the LC/MS conventional methodology can be cited as the main advantages of the on line coacervation with fluorescence detection proposed methodology.

Keywords: Flow Injection Analysis; Fluorescence Detection; Bile Salt Coacervation; LCarnitine; Pharmaceutical Quality Control. 2

ACCEPTED MANUSCRIPT 1. Introduction Carnitine (γ-trimethyl-β-hydroxybutyrobetaine, CAR) is an endogenous metabolite

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found in most mammals. It is synthesized in tissues such as brain, liver, and kidney from

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the amino acids lysine and methionine [1-4]. CAR is involved mainly in depleting the

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formation of free radicals in various tissues, maintaining the cellular concentration of free coenzyme A [5], eliminating potentially toxic compounds, and transporting long chain fatty

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acids to the mitochondrial or peroxisomal matrix for its β-oxidation [6, 7]. Skeletal muscle

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is the major reservoir of CAR (approximately 95%) under its biological active form, LCAR, and approximately three quarters of this reserve comes from the diet (mainly meat

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and derivatives and, more recently, from dietary supplements) [8]. Based on nutritional

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studies carried out in humans, it has been proposed that the daily need for L- CAR is between 2 and 12 µmol kg-1 (0.3 to 1.9 mg kg-1) of body weight day-1. However, these

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amounts are higher in athletes or pregnant women [9]. Deficiency of this metabolite is related to several pathologies including dyslipoproteinemia, anorexia, dyspepsia, cardiac arrhythmias, erectile dysfunction, and others [10, 11]. In this context, the development of new, fast and simple methods for routine determination of CAR is of great interest for the quality control of dietary supplements and other dosage forms. To date, there is available a number of methods for CAR determination, and most of them involve high performance liquid chromatography or capillary electrophoresis coupled to tandem mass detection or UV direct or indirect detection [7,12-14]. On the other hand, several chromatographic techniques with fluorescent detection has been developed for L-CAR and related compounds [15-17] Examples are found in the US Pharmacopoeia [18], and European Pharmacopoeia [19].

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ACCEPTED MANUSCRIPT Also, electrochemical methods have been proposed [8, 20, 21]. More recently, the use of flow injection (FI) strategies with acid hydrolysis have been assayed in association with

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tandem mass spectrometry for quantification of plasma free and total CAR. Also a strategy

through the NADH fluorescence detection [22, 23].

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by L-carnitine dehydrogenase enzyme was used for indirect determination of the L-CAR

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CAR does not present fluorescent emission, and therefore for molecular fluorescence

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detection it is necessary a pre-derivatization step with a fluorophore. Antecedents available in bibliography show that 9-fluorenylmethyl chloroformate (FMOC) is an adequate reagent

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to enhance UV sensitivity of carnitine (Fig. 1), but also to give fluorescent activity to this compound [24].

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As an alternative to HPLC methods, FI schemes could be designed to offer a significant

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improve in the sampling throughput, economy of the sample and reagents, minimizing the

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wastes generated; furthermore, FI schemes are easy to couple with different optical detectors. The experimental setup usually involves selecting an extraction/retention agent, and an adequate interfacing manifold [25]. In this scenario, surfactant mediated extractions (SME) have become popular to enrich and/or recover biological compounds from matrices of different nature [26]. Despite most SME are performed in batch mode, there is an increasingly number of reports dealing with their utilization in flow injection analysis (FIA) formats [27-29]. On-line SME offers advantages in terms of better sensitivity and precision as well as of a significant reduction of the amounts of the sample and reagents required and wastes generated. In some of these applications, high temperature was required to induce phase separation. However, elevated temperature, in addition of the experimental difficult that represent, may affect the stability of thermally labile analytes.

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ACCEPTED MANUSCRIPT Bile salts (BS) are biosurfactants that are synthesized from cholesterol in the liver. BS micelles, such as sodium taurodeoxycholate (NaTDC), have unique properties including

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that it does not foam and scatter light; and other features and characteristic that have been

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discussed in a previous work In the previous report, the bile salt sodium cholate was evaluated as coacervate agent for the preconcentration/separation of the coloring dye

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Rhodamine B in food condiments [30]. Aqueous solutions of BS, such as NaTDC, can form

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coacervate phases in the presence of additives such as quaternary ammonium surfactants, alcohols or acids. BS have been used in coacervation processes at room temperature and

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successfully applied to varied analytes [31, 32].

The aim of this work was to develop a new method for L- CAR determination in dietary

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supplements. This is an alternative method that uses, for the first time, a flow injection

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scheme that joins the extraction efficiency and specificity of NaTDC towards the L- CAR -

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FMOC derivative, with the reproducibility and sensitivity of molecular fluorescence (MF). The study focused on sensitivity and spectrofluorimetric characteristics at first, which involves experimental conditions for derivatization of L- CAR with FMOC and reaction conditions for coacervate extraction. To this aim, a batch scheme was employed. In a second stage, the extraction/enrichment characteristics were suited to on-line process pointing not only to reduce reagent and sample consumption, but also to enhance sensitivity and sure reproducibility. The conditions studied were the sample loading flow rate, the loading times and volume, and the elution agents, concentrations and time in an on-line manifold to form the FI extraction system. After optimization, this proposed method was applied for determination of L- CAR in commercially available dietary supplements samples, for quality control.

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ACCEPTED MANUSCRIPT 2.

Materials and methods

2.1 Chemicals and Reagents

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L-Carnitine (L- CAR) inner salt, 98% was purchased from Sigma-Aldrich (St. Louis,

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USA). 9-fluorenylmethyl chloroformate (FMOC) and Sodium taurodeoxycholate were

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obtained from (Sigma Chemical Co., St. Louis, MO, United States). Absolute ethanol, hydrochloric acid, acetonitrile and sodium carbonate were purchased from (Merck,

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Darmstadt, Germany), were acquired as indicated and used as received without further

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purification. Ultrapure water (18 MΩ cm) was obtained from EASY pure (RF Barnstead,

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IA, USA). All other reagents employed in this study were of analytical grade quality.

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2.2 Instrumentation

A Shimadzu RF-5301PC spectrofluorimeter (ShimadzuCorporation, Analytical Instrument

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Division, Kyoto, Japan), equipped with a Xenon discharge lamp and a photomultiplier tube (PMT) voltage was 600 V was used for the fluorescent measurements. A 1.0 cm quartz cell was employed for the batch assays and a 120 L flow cell unit (Shimadzu Corporation, Analytical Instrument Division, Kyoto, Japan) for the flow measurements was used. LCAR derivatized with FMOC was analyzed operating the spectrofluorimeter in the timecourse mode (transient signals; λex: 266 nm; λem: 313 nm, slits: 5/5 nm). The FIA manifold consisted of a Rheodyne (Rohnert Park, CA) model-5020 six-port two-way rotary valve. Standard and sample solutions were pumped through the FIA system with two Gilson (Villiers, France) Minipuls 2 peristaltic pumps connected with 1.3 mm i.d. Tygon tubing (Middleton, WI, USA).

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ACCEPTED MANUSCRIPT 2.3 Stock solutions L-Car stock solutions 6.2x10-4 mol L-1 were prepared by dissolution of suitable amounts in

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ultrapure water. Similarly, 0.020 mol L-1 NaTDC solution was prepared by dissolution of

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the solid surfactant in ultrapure water. A 1.0 mol L-1 HCl solution was prepared by dilution

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of concentrated HCl with ultrapure water. 2.4 Derivatization procedure

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The following solutions were prepared for use in the derivatization procedure according to

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[24]: An aliquot of standard L- CAR 6.2x10-4 mol L-1 (50 µL) was mixed with 50 µL of carbonate buffer (50x10-3 mol L-1, pH 10.4) and 130 µL of FMOC (30x10-3mol L-1 in

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acetonitrile), were allowed to react for 60 min at 50 °C. The reaction was stopped with 150

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µL acetate buffer (50 x10-3mol L-1, pH 4.2).

2.5 Samples preparation

Samples of dietary supplements, drink and injectable, were acquired in local shops and pharmacies. Different aliquots of dietary supplements were diluted in ultrapure water to achieve a final concentration similar to L- CAR standard solution and homogenized for 5 minutes using a magnetic stirrer. Then, the derivatization procedure was performed, and 100 µL of the resulting solution were diluted up to 5 mL with ultrapure water and ethanol (80:20). An aliquot (1 mL) of the final dilution was then added to 1.0 mL NaTDC (0.020 mol L-1), 300 µL of HCl (1.0 mol L-1) and made up to 10 mL final volume with ultrapure water.

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ACCEPTED MANUSCRIPT 2.6 On-line extraction and fluorescence detection The analytical procedure proposed is very simple and it could be realized by mean a unique

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peristaltic pump. However, with the aim of optimize as independent parameters Sample

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Flow Rate (SFR) and Eluent Flow Rate (EFR), the FI system was designed using two peristaltic pumps (see Fig. 2). Once prepared, the resulting coacervate containing the

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analyte was analyzed using an on-line phase separation/preconcentration system that

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included a packed mini column coupled to the flow cell of a spectrofluorimeter (Fig. 2). The L- CAR -FMOC derivate (S) prepared as just noted, were loaded on to the glass wool

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mini column (mC) that was connected between ports 1 and 4 of the six-port valve (V) in position a (loading). In this step, L- CAR -FMOC derivate coacervate contained was

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retained while the aqueous phase was sent to waste. Simultaneously, the eluent phase (0.01

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mol L-1 HCl solution) flowed through the line E to the detector to record the baseline

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fluorescence. Finally, the sample peristaltic pump (P1) was stopped, the valve V was switched to injection position (b), and L- CAR -FMOC derivate coacervate contained was eluted with 0.01 mol L-1 HCl solution, directly into the flow cell. The analytical signal was recorded as peak area which was proportional to the L- CAR concentration in the sample. Afterwards, valve was switched to load position and the sequence started again for the next sample.

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Results and discussion

3.1. Surfactant mediated extraction variables The main goal of this study was to find the best conditions for fast and quantitative extraction of L- CAR -FMOC derivate. Sodium taurodeoxycholate was evaluated as

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ACCEPTED MANUSCRIPT coacervate agents in the concentration range of 1.0x10-3 to 4.0x10-3 mol L-1. The maximum extraction efficiency was found to be at 2.0x10-3 mol L-1, therefore this value was selected

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for further studies. Based on previous studies (results not shown here), several coacervate

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promoting agents were assayed in aqueous media [29]. Thus, variable concentrations of calcium chloride, potassium iodide, sodium chloride, hexadecyl trimethyl ammonium

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bromide (HTAB), sodium dodecylsulfate (SDS) and HCl were added to aqueous solutions

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of NaTDC containing L- CAR -FMOC, and solutions evaluated for signs of coacervate formation. The ranges of concentrations studied were 0 - 0.05 mol L-1 for calcium chloride,

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potassium iodide and HCl. While for SDS and HTAB the concentration range studied were 1.0x10-3 – 3.0x10-3 mol L-1 and 0 – 5.0x10-4 mol L-1; respectively. Conclusions about

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efficiency of extraction and ease of phase separation were laid out through comparison of

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the analytical signal recorded after phase separation and fluorescence measurement. The

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inference of this study indicated that better results, in terms of intensity fluorescent signals, were obtained using HCl as coacervate promoting agent.

3.2. On-line preconcentration/Phase separation The HCl concentration required for retention of the formed coacervate on the glass wool in the mC was re-optimized using on-line scheme, considering the maximum fluorescence signal and the adequate repeatability. The obtained results were in concordance with the batch studies. Some batch studies were conducted in order to determine the optimal concentrations of HCl required for retention of the formed coacervate on the glass wool into C (as gauged by its physical appearance) and for obtainment of the maximum L- CAR -FMOC fluorescence signal. The influence of HCl volume on the coacervate formation was evaluated in the 9

ACCEPTED MANUSCRIPT range 0- 0.05 mol L-1. The best results were obtained with 2.0x10-3 mol L-1 of NaTDC and 0.03 mol-1 L-1 of HCl. All subsequent experiments were conducted using these conditions.

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Column design, packing material and packing conditions were also evaluated. The packings

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analyzed were: HPLC filter, glass wool, native multiwall carbon nanotubes derivatized with NaTDC and cyclodextrins. Results showed that the minicolumn filled with 25 mg of glass

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wool was sufficient for the quantitative retention of the NaTDC coacervate containing the

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3.3. Sample loading and elution variables

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L-Car- FMOC and for generating maximal and reproducible signals.

The variables influencing the performance of the FIA method were examined to determine

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the conditions necessary for an optimal fluorescence signal. First, the influence of SFR on

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the sensitivity was evaluated between the range of 0.5 and 3.0 mL min-1. A signal decrease

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was observed at sample flow rates above 2.0 mL min-1. Reasonable explanation to this observation is that analyte was retained partially due to insufficient contact time with the solid phase. Therefore, a SFR of 2.0 mL min-1 was selected as optimal. In second term, the eluent composition and elution flow rate were optimized. Different solvents, such as water, ethanol, acetonitrile, different acid buffer solutions, HCl aqueous solutions and mixtures of them were tested as eluents to achieve complete release of the analyte from the NaTDC coacervate with optimal analytical signal. Weak fluorescence signals (evaluated as peak area) were obtained when water, ethanol, acetonitrile, sodium acetate acetic acid buffer (pH 4.0); water: ethanol or water: acetonitrile mixtures were employed as the eluent solvent systems. However, satisfactory elutions were achieved when solutions of HCl were employed as eluent. Hence, HCl was selected as eluent and the effect of HCl concentration was optimized from 1.0x10-3 to 1.0 mol L-1. The optimal concentration considering the 10

ACCEPTED MANUSCRIPT quantitative elution and adequate sensitivity for L- CAR -FMOC was found to be 1.0x10-2 mol L-1 of HCl. Moreover, the effect of EFR on the analytical response was studied at flow

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rates between 1.0 and 3.0 mL min-1. The results showed that the analytical signal increased

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with the EFR, but at flow rates greater than 3.0 mL min-1 large and undesirable backpressures touching off uncouple on the FI system occurred. Therefore, an elution flow rate

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of 2.5 mL min-1 was selected as optimal because it allowed reasonable analysis time and

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acceptable mini-column back pressure. Furthermore, considering the reported in previous works [30], the influence of the direction elution flow on the sensitivity and peak shape was

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studied. The best results were obtained when the elution was performed in a countercurrent

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3.4. Analytical performance

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manner (Fig. 2).

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The calibration plot of FIA fluorescence peak area vs. L- CAR - FMOC concentration was linear over the concentration range (limit of linearity, LoL) of 3.26x10-7– 2.60x10-6 mol L-1 L- CAR - FMOC (r2= 0.9932). The limits of detection (LoD) and quantification (LoQ) were calculated using the relation k (SD)/m where k= 3.3 for LoD and 10 for LoQ and SD represents the standard deviation from 15 replicate blank responses and m is the slope of the calibration curve [33]. The LoD and LoQ obtained values were 2.4x10-8 mol L-1 and 7.3x10-8 mol L-1, respectively. A comparison of the sensitivity of this method with other reported methods in the literature [8, 13, 20, 21, 23, 34] is presented in Table 1. The present method offers simplicity, robustness and high sample throughput (30 samples per hour, without consider the required time for L-CAR derivatization) compared to previously reported methodologies. In addition, the achieved analytical parameters were similar to the informed by other authors. 11

ACCEPTED MANUSCRIPT 3.5. Application to real samples The L- CAR extraction using NaTDC coupled to molecular fluorescence was first applied

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to the L- CAR quantification in dietary supplements commercialized in different

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pharmacies of San Luis city, Argentina. The Fig. 3 shows the transient responses obtained for the standards and a real sample analyzed using the recommended optimized procedure.

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As can be seen in the Table 2, the L- CAR found in two of the samples analyzed were

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according to the percent specified in the corresponding monograph [18]. On the other hand, the recovery obtained for L- CAR -FMOC in the dietary supplements

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(drink and injectable) spiked with standard solutions at three concentration levels (8.0x10-8, 1.63x10-7 and 2.44x10-7 mol L-1 of L- CAR -FMOC) were satisfactory (recoveries between

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82 % and 103 % were obtained). These outcomes showed that no matrix interferences were

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detected at none of the analyzed samples. Moreover, the L- CAR found concentration was

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statically in agreement with the labels of pharmaceutical formulations.

4. Conclusions

An on-line surfactant-mediated extraction method using the bile salt NaTDC, as the coacervate agent, with fluorimetric detection for determination of L- CAR in dietary supplements has been developed. The proposed method reported for the first time the use of bile salt taurodeoxycholate, surfactant of natural origin, as a clouding phenomenon promoter agent. The automation of the methodology was achieved by employing a scheme of flow injection analysis, obtaining a sample throughput of 30 samples per hour (without consider the required time for L-CAR derivatization). The developed method resulted to be simple, fast, reliable, and economic, showing low detection limits compatible with the 12

ACCEPTED MANUSCRIPT concentration of this compound in complex matrix as pharmaceutical formulations. Moreover, the proposed methodology represents a viable alternative to the conventional

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carnitine analysis methods, as well as being environmentally friendly.

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Acknowledgments

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The authors wish to acknowledge the financial support received by Agencia Nacional de Promoción Científica y Tecnológica, Project PICT- 2010- 189, Instituto de Química de San

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Luis (INQUISAL-CONICET), Project PIP 11220130100605 and Universidad Nacional de

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San Luis, Project PROICO 22/Q228 and 22/Q832.

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ACCEPTED MANUSCRIPT Figure Captions

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Figure 1: Chemical structures of L-Car derivatized with FMOC and sodium

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taurodeoxycholate (used as coacervate agent) under the experimental conditions employed.

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Figure 2: Schematic diagram of the on-line extraction approach mediated by NaTDC for

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L-Car determination, mC, minicolumn; PP, peristaltic pumps; V, load/injection valve: (a)

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loading position (b) inject position); D, spectrofluorimeter.

Figure 3: Transient signal (FIAgram) obtained under optimized conditions corresponding

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to both L-Car standard calibration and sample signal. Standard solution of L-Car-FMOC:

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(a) 3.26x10-7 mol L-1; (b) 9.78 x10-7 mol L-1; (c) 1.30 x10-6 mol L-1; (d) 1.95 x10-6 mol

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L-1; (e) 2.6 x10-6 mol L-1; (f) real sample (Albicar inyectable). [λex = 266 nm and λem = 313nm. Excitation and emission slit widths were 5 nm and 5 nm, respectively. Injection time: 40 s.

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Figure 1

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Figure 2

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Figure 3

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Table 1. Comparative study of the proposed method with others reported in the literature for the determination of L-carnitine in dietary

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supplements.

Figures of Merit

Capillary Electrophoresis with contact less

LoD: 2.6x10-9 mol L-1, LoQ: 7.9x10-9 mol L-1, Analysis Time: 4

conductivity detection

min, Recovery: 93.5± 5.7 – 102± 3.4 %

ITP-ITP

LoD: 3.1x10-6 mol L-1, LoQ: 1.2x10-5 mol L-1, Analysis Time: 25

Reference [8]

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Methodology

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min, Recovery: 91 – 113 %

LoD: 2.6x10-5 mol L-1, LoQ: 8.7x10-5 mol L-1, Analysis Time: 10 [13]

LoD: 2.7x10-5 mol L-1, LoQ: 9.11x10-5 mol L-1, Analysis Time: 10

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Direct CZE-UV

Ion Selective Electrodes

min, Recovery: 104- 106 %

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Indirect CZE-UV

min, Recovery: 96- 98%

LoD: 2.7x10-5 mol L-1 (DOS), 2.1x10-5 mol L-1 (TCP), 6.8x10-6 mol

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L-1 (NPOE), Analysis Time: ˂ 5 seg. Ion Pair Chromatography with indirect

LoD: 1.7x10-5 mol L-1, Analysis Time:

conductimetric detection

˃5.4 min, Recovery: 97.7 – 99.7 %

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LoD: 1.0x10-6 mol L-1, Analysis Time: 4.2 min.

LC-MS/MS

LOD: 3.1x10-10 mol L-1, LoQ: 1x10-9 mol L-1, Analysis Time: 20

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FIA with NADH Fluorescence Detection

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min, Recovery: 93.38 % LoD: 2.4x10-8 mol L-1, LoQ: 7.3x10-8 mol L-1, Analysis Time: 2

NaTDC coacervate coupled to Molecular

min, Recovery: 82 – 103 %

This work

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FIA method using extraction mediated by

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Fluorescence

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Table 2. Labeled concentration vs found concentration of L-CAR in dietary supplements

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(drink and injectable commercial presentation forms) analyzed by Flow Injection Analysis

Labeled L-CAR

Found L-CAR ± SD

Recovery (%)

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1g/10 mL

0.97± 0.02 g/10 mL

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250 mg/7.5 g

271±3.79 mg/ 7.5 g

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1g/5 mL

0.84±0.04 g/5mL

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coupled to Molecular Fluorescence

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Sample*

1- Drink Albicar™ (10 µL/ 10 mL), composition: sodium cyclamate, hydrochloric acid, potassium

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sorbate, cetrimide, ethanol, orange flavor, sodium saccharin and distilled water, 2- Drink

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Omnilife™ (7,2 mL/100 mL), composition: sweetener, green coffee extract, black tea, inuline, LTyrosine and preservatives, 3-Injectable Albicar™ (5 µL/10 mL), composition: hydrochloric acid and distilled water sterile .

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ACCEPTED MANUSCRIPT Highlights Novel method has been developed for L-Carnitine fluorescent quantification.

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On line sodium taurodeoxycholate coacervate mediated extraction has been applied.

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It constitutes an alternative strategy to L- Carnitine conventional analysis.

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It is a green method considering the low volume consumption of reagents.

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