Determination of hippuric acid in biological fluids using single drop liquid–liquid-liquid microextraction

Abstract

A simple, sensitive, and inexpensive single drop liquid–liquid-liquid microextraction (LLLME) followed by isocratic reverse phase high performance liquid chromatography (RP-HPLC) and UV detection was developed for determination of hippuric acid in human urine and serum samples. The analyte was extracted from an acidic aqueous sample solution (pH 3) through a thin layer of organic solvent membrane and back-extracted to a basic acceptor drop (pH 11) suspended on the tip of a 10-μL HPLC syringe in the organic layer. The influence of several important parameters on extraction efficiency of hippuric acid was evaluated. Under optimized experimental conditions, the calibration graph was linear in the concentration range of 1–400 μg L⁻¹ with coefficient of determination 0.998. The limits of detection and quantification were 0.3 and 1.0 μg L⁻¹, respectively. Intra-day and inter-day precision were in the range of 1.1–2.7% and 1.1–3.1%, respectively. This procedure was successfully applied to the determination of hippuric acid in spiked urine and serum samples with satisfactory results. The relative recoveries of urine and serum samples ranged from 91.4 to 99.3%, with relative standard deviations varying from 1.6 to 4.2%.

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Introduction

Toluene is one of the most important industrial organic chemicals. It is absorbed by humans through the lungs, and more than 80% of the toluene absorbed is metabolized to hippuric acid.^1–4 Those who are exposed to toluene for long times have been found to suffer from anatomical changes in the brain and neurobehavioral impairments.⁵ Hippuric acid, a colorless crystal, is one of the most biological indicators of occupational exposure to toluene, polyhydrophenols, glycine, and benzoic acid.^5–9 Hippuric acid may also derive from alkyl benzenes (widely used as organic solvents in industry), styrene (widely used in the plastics industry and has been implicated as a reproductive toxicant and possible carcinogen), food containing sodium benzoate (added as a preservative), phenylalanine, other alkylbenzenes, or fine aerosol engine emissions.^10–12 Hippuric acid is excreted in the human urine.^9,13–16

Some sample preparation methods have been reported for the pre-treatment of hippuric acid in urine samples. Liquid–liquid extraction (LLE)¹⁵ and solid phase microextraction (SPME)^11,17 are two of the most useful sample preparation methods. The main drawbacks of LLE are that it is tedious, time-consuming, has low sensitivity, and needs large amount of toxic organic solvents. On the other hand SPME needs considerably less volume of solvent compared to LLE method, but it is relatively expensive, its fiber is fragile and has limited life-time and sample carry-over can be a problem.¹⁸ Therefore, a simple, fast, and more efficient method is necessary for extraction and quantification of hippuric acid in biological samples.

Hippuric acid is a weak acid (pK_a 3.62). It can be extracted from an acidic aqueous solution into a basic aqueous phase by single drop liquid–liquid-liquid microextraction (LLLME). LLLME involves a series of two reversible extractions. In three phases LLLME analytes are first extracted into a thin layer organic membrane phase (in their neutral form) from an aqueous donor phase and second back-extracted into an aqueous acceptor phase.^19,20 LLLME is a simple, fast, and inexpensive technique. In this method, high preconcentration may be achieved. LLLME uses minimal amounts of solvent that enables extraction and concentration steps to be carried out simultaneously. The greatest feature of this method is excellent clean-up that enables the extraction of analytes from complex matrices such as biological fluids.^21–23

In this work a simple, rapid, sensitive, and inexpensive LLLME method has been developed for extraction of hippuric acid in human urine and serum samples then RP-HPLC method with UV detection was used for quantification of analyte. To the best of our knowledge, LLLME method has not been previously reported for the determination of hippuric acid in biological fluid samples.

Experimental

Chemicals and solvents

All chemicals were analytical HPLC reagent grade and used as received. Cyclohexane, ethyl acetate, o-xylene, acetonitrile, water, phosphoric acid (H₃PO₄), and sodium hydroxide (NaOH) were supplied by Merck (Darmstadt, Germany). 2-Ethyl-1-hexanol was purchased from Riedel-de Haen (Germany). Hippuric acid (N-benzoylglycine, 98%) and butyl acetate were supplied from Acros (Geel, Belgium).

HPLC analysis

The HPLC system (model SCL-10Avp, Shimadzu, Japan) consists of a UV detector (model SPD-10Avp), operating at wavelength of 228 nm, dual solvent pump (model LC-10Avp), and an injection valve (model EIG 001). The analytical isocratic RP-HPLC separation was performed on a Shim-pack CLC-ODS-C8 column (6 × 150 mm, particle size, 5 μm) with a guard column (CLC G-ODS). The mobile phase was made up of acetonitrile : H₃PO₄ buffer (10 : 90, v/v) adjusted to pH 3.0 with 1.0 mol L⁻¹ sodium hydroxide; the flow rate was 1 mL min⁻¹.

LLLME procedure

The schematic diagram of the LLLME setup may be found in the literature.²⁴ In each extraction, 5 mL of aqueous sample solution (adjusted to pH 3 with 0.02 mol L⁻¹ H₃PO₄) containing analyte was taken into a 5-mL volumetric flask. A 300-μL portion of organic phase was added to the top of aqueous phase via an Eppendorf micropipette sampler. A 10-μL syringe was used for suspending the microdrop (7 μL, pH was adjusted to 11 with 0.5 mol L⁻¹ NaOH) to the organic phase during extraction and also for injection into the HPLC injection valve after extraction. A magnetic stirring bar (8.5 mm × 3.0 mm) was placed into the solution to provide sufficient stirring extraction. After 45 min, the microdrop was retracted back into the syringe and immediately transferred to the injection valve of HPLC system for analysis.

Preparation of standard reference samples

Stock solution of hippuric acid was prepared in double distilled water (pH 3) at the concentration of 50 mg L⁻¹. Working solutions were obtained by appropriate dilution of the stock solution. Calibration standards were made at different concentration ranges. Each one was prepared in three replicates. Equation was obtained by least squares linear regression of the peak area versus analyte concentrations. A solution 100 μg L⁻¹ hippuric acid was used for optimization of LLLME procedure. All solutions were stored at 4 °C in the dark.

Urine and serum samples

Urine and serum samples were kindly donated by volunteers. The urine sample was filtered using Whatman No. 42 filter paper and centrifuged before analysis. The sample was processed immediately or stored at 4 °C in the dark. For analysis, an aliquot of 1 mL of urine sample was transferred to a 25-mL volumetric flask diluted up to volume with double distilled water. Then, 1 mL of this solution was transferred into a 5-mL volumetric flask, fortified with analyte, diluted up to volume with double distilled water (pH 3), and submitted to LLLME-HPLC-UV.

Serum sample (5 mL) was mixed with 3 mL acetonitrile and shaken vigorously. After 1 min, the sample was centrifuged for 5 min at 4000 rpm, and processed immediately or stored at 4 °C in the dark. A 1-mL of supernatant was transferred to a 5-mL volumetric flask, fortified with analyte, diluted up to volume with double distilled water (pH 3), and submitted to LLLME-HPLC-UV.

Results and discussion

In this study the effects of several important parameters influencing the extraction efficiency of hippuric acid including type and volume of organic solvent, volume of acceptor aqueous phase, composition of donor and acceptor phases, stirring rate, and extraction time were investigated.

Effect of type and volume of organic solvent

One of the most important steps in the development of the proposed method is the selection of appropriate organic solvents. The selection of organic solvents was based on (a) lower density than water (b) immiscibility with water (c) low volatility and (d) solubility of target compound in organic solvents. Based on these considerations, five water-immiscible organic solvents with different characteristics including, cyclohexane, o-xylene, butyl acetate, ethyl acetate, and 2-ethyl-1-hexanol were tested (Fig. 1). The results demonstrated that butyl acetate : ethyl acetate (1 : 2, v/v) provided higher extraction efficiency than other solvents. Therefore, butyl acetate : ethyl acetate (1 : 2, v/v) was selected as the organic phase for LLLME.

Fig. 1 Effect of the organic solvents on the extraction efficiency of hippuric acid. Extraction conditions: donor phase volume = 5 mL aqueous solution (pH 3); acceptor phase volume = 5 μL of aqueous solution (pH 10); extraction time = 45 min; stirring rate = 500 rpm; volume of organic phase = 300 μL. The organic solvents are (A), cyclohexane; (B), o-xylene; (C), 2-ethyl-1-hexanol; (D), butyl acetate; (E), ethyl acetate; (F), butyl acetate + ethyl acetate (2 : 1, v/v); (G), butyl acetate + ethyl acetate (1 : 1, v/v); (H), butyl acetate + ethyl acetate (1 : 2, v/v).

The effect of volume of selected organic phase was investigated in the range 100–400 μL. From the results of Fig. 2, it can be seen that an increase in the volume of organic phase increased the extraction efficiency in the range of 100–300 μL. With less than 100 μL of organic solvent, the organic layer was thin, the acceptor phase was unstable. However, a further increase in the volume of organic phase followed by a decrease in peak area of analyte. This may be because of greater dilution of the extract. Therefore, 300 μL was found to be optimum for the following experiments.

Fig. 2 Effect of volume of organic phase on the extraction efficiency. Extraction conditions: donor phase volume = 5 mL aqueous solution (pH 3); acceptor phase volume = 5 μL of aqueous solution (pH 10); extraction time = 45 min; stirring rate = 500 rpm.

Effect of volume of acceptor phase

To increase the sensitivity of analytical signal, the acceptor phase volume was studied in the range 3–9 μL using 0.5 mol L⁻¹ NaOH (pH 10). However, the acceptor phase volumes greater than 9 μL were unstable when suspending at the tip of the microsyringe. The results demonstrated in Fig. 3 reveals that the analytical signal initially increases with an increase of the acceptor phase volume up to 7 μL, followed by a decrease in peak area with further increase in the acceptor phase volume. On the basis of the above results, 7 μL was used as the optimum acceptor phase volume for subsequent experiments.

Fig. 3 Effect of volume of acceptor phase on the extraction efficiency. Extraction conditions: donor phase volume = 5 mL aqueous solution (pH 3); extraction time = 45 min; stirring rate = 500 rpm; volume of organic phase = 300 μL; pH of acceptor phase = 10.

Composition of donor and acceptor phases

Because pH value of donor and acceptor phases play a unique role in LLLME method and subsequent extraction efficiency, the pH value of donor and acceptor phases were investigated for its effect on the extraction of hippuric acid. The donor phase should be sufficiently acidic in order to deionize hippuric acid and consequently reduce its solubility in the donor phase. The pH value of the donor phase was varied in the range of 2–6 using different concentrations of H₃PO₄ (Table 1). The experimental results demonstrated that the signal was increased by increasing the pH up to 3 and followed by decreasing with further increasing pH of donor phase. The results are presented in Fig. 4. The pH value of the acceptor phase should be sufficiently alkaline that promotes the dissolution of hippuric acid, which prevents the analyte from re-extracting the organic phase. The effect of pH of acceptor phase was investigated in the range 8–13 using different concentrations of NaOH solution (Table 1). It was found that, for the acceptor phase pH value of 11 was the best choice for extraction of analyte (Fig. 4). Based on the above results, pH values of 3 and 11 were selected for the donor and the acceptor phases, respectively.

Table 1 Effect of composition of donor and acceptor phases on the extraction efficiency of hippuric acid (n = 3)^a

	H3PO4/0.02 mol L^−1			NaOH/0.5 mol L^−1
	0.1/mol L^−1 NaOH	0.5/mol L^−1 NaOH	1.0/mol L^−1 NaOH	0.01/mol L^−1 H3PO4	0.02/mol L^−1 H3PO4	0.05/mol L^−1 H3PO4
a The extraction conditions: donor phase volume =5 mL; acceptor phase volume = 7 μL; organic phase volume = 300 μL; extraction time = 45 min; stirring rate = 500 rpm.
Peak area	168041.7	168042.7	168042.0	168040.6	168044.0	168042.3

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Fig. 4 Effect of pH of donor and acceptor phases on the extraction efficiency. Extraction conditions: donor phase volume = 5 mL; acceptor phase volume = 7 μL; extraction time = 45 min; stirring rate = 500 rpm; volume of organic phase = 300 μL.

Effect of stirring rate

Stirring rate reduces the time required to reach thermodynamic equilibrium and increases the extraction efficiency. Faster stirring rate could be employed to improve the extraction efficiency, since agitation permits the continuous exposure of the extraction surface to fresh aqueous samples.^25–27 The effect of stirring rate on the extraction efficiency was studied in the range 100–800 rpm (data not shown). The results indicated that the extraction efficiency increased with increasing of stirring rate from 100 to 700 rpm. Due to instability of microdrop, stirring rate above 700 rpm was not evaluated. Therefore, all further experiments were performed with a stirring rate of 700 rpm.

Effect of extraction time

Since LLLME is an equilibrium extraction procedure, the maximum amount of analyte can be extracted by the acceptor phase after equilibrium is obtained. Effect of extraction time on the extraction efficiency was examined in the range of 15–55 min at optimized experimental conditions. As shown in Fig. 5, the extraction efficiency increased with increasing extraction time from 15 to 45 min and reached equilibrium at 45 min. After 45 min, the curve reached a plateau and no increase was observed with additional time. Therefore, an extraction time of 45 min was selected for subsequent experiments.

Fig. 5 Effect of extraction time on the extraction efficiency. Extraction conditions: donor phase volume = 5 mL aqueous solution (pH 3); acceptor phase volume = 7 μL of aqueous solution (pH 11); stirring rate = 700 rpm; volume of organic phase = 300 μL.

Method validation

Under the above optimum experimental conditions, the proposed method was validated by linearity, limits of detection and quantification, enrichment factor, precision, and accuracy. The calibration plot was found to be linear in the range of 1–400 μg L⁻¹, with a coefficient of determination (r²) of 0.998 (n = 9). For each concentration level, three replicate extractions were performed.

The limits of detection (LOD, S/N = 3) and quantification (LOQ, S/N = 10) of hippuric acid were 0.3 and 1.0 μg L⁻¹, respectively. As it can be seen, the proposed method has low LOD and can be used for trace analysis of hippuric acid in biological fluid samples.

The enrichment factor was defined as the ratio of the peak area after LLLME method to the peak area of standard solution at the same concentrations.^28,29 The enrichment was 257.1 for hippuric acid in aqueous sample containing 200 μg L⁻¹.

The intra-day and inter-day precision of the proposed method were calculated by analyzing replicate (n = 5) urine and serum samples spiked with three different concentration levels (5, 50, and 200 μg L⁻¹) of hippuric acid (Table 2). As it can be seen from Table 2, the intra-day and inter-day precisions in urine were in the range of 1.1–2.3% and 1.1–2.6%, respectively. The intra-day and inter-day precisions in serum were in the range of 1.1–2.7% and 1.2–3.1%, respectively.

Table 2 Intra-and inter day precisions of hippuric acid analysis in urine and serum samples by standard addition method (n = 5)

Sample	Add/μg L^−1	Intra-day		Inter-day
		Found (mean ± SD)^a	%RSD	Found (mean ± SD)^a	%RSD
a Results were expressed as μg L^−1.
Urine	5.0	4.8 ± 0.11	2.3	5.0 ± 0.13	2.3
	50.0	51.0 ± 1.10	2.1	49.3 ± 1.30	2.6
	200.0	198.0 ± 2.06	1.1	198.5 ± 2.20	1.1
Serum	5.0	4.6 ± 0.10	2.2	4.7 ± 0.13	2.8
	50.0	47.3 ± 1.30	2.7	48.0 ± 1.50	3.1
	200.0	196.5 ± 2.1	1.1	195.5 ± 2.30	1.2

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In order to determine the accuracy and the extraction recovery of the proposed method, the standard addition test was performed. In which, the standard solutions of target compound were prepared with different concentration levels. Three standard solutions of different concentration levels were added to known volume of urine and serum samples, respectively. The resultant samples were extracted with the proposed method and analyzed. Three replicate extractions were performed for each concentration level, and the ratio of measured and added amounts was used to calculate the extraction recovery. The results are summarized in Table 3. The results show that the recoveries, measured at three concentration levels, varied from 91.4 to 99.3% with relative standard deviations (RSDs) less than 4.2%.

Table 3 Results from determination of recovery of hippuric acid in urine and serum samples by standard addition method (n = 3)

Sample	Added/μg L^−1	Found (mean ± SD)^a	%RSD	%Recovery
a Results were expressed as μg L^−1.
Urine	0.0	0.8 ± 0.02	2.5	—
	7.0	7.6 ± 0.12	1.8	97.1
	35.0	35.2 ± 0.60	2.9	98.3
	150.0	149.7 ± 2.34	1.6	99.3
Serum	0.0	0.5 ± 0.01	2.0	—
	7.0	6.9 ± 0.17	3.1	91.4
	35.0	33.7 ± 1.40	4.2	94.3
	150.0	143.7 ± 4.41	3.1	95.5

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To test the applicability of the proposed LLLME method in real sample analysis, the determination of hippuric acid in urine and serum samples were performed. Representative chromatograms of different sample extracts are shown in Fig. 6. As it can be seen, no significant interference peaks were found at the retention position of analyte.

Fig. 6 HPLC-UV chromatograms of hippuric acid obtained by LLLME under optimized conditions: (a) blank serum; (b) spiked serum with 10 μg L⁻¹; (c) blank urine, and (d) spiked urine with 10 μg L⁻¹. Peak: (HA) Hippuric acid.

Table 4 indicates the limit of detection (LOD), linear range (LR), relative standard deviation (RSD), and recovery, using LC-MS/MS,³⁰ gas chromatography-mass spectrometry,¹⁵ HPLC,³¹ HPLC-MS,³² solid phase microextraction and gas chromatography-ion trap tandem mass spectrometry,¹¹ empore disk and gas chromatography-mass spectrometry,³ and single drop liquid–liquid-liquid microextraction and-high performance liquid chromatography-UV detection (LLLME-HPLC-UV) methods for the determination of hippuric acid in urine sample.

Table 4 Comparison of LLLME method with reported methods for the determination of hippuric acid in urine matrix

Parameter	Value/and remark
	Reported method	LLLME
a Ref. 30. b Ref. 15. c Ref. 31. d Ref. 32. e Ref. 11. f Ref. 3.
LOD	17/pg^b; 4.9/mg L^−1^c; 5/ng mL^−1^d; 630/μg L^−1^e; 2.5/μg mL^−1^f	0.3/μg L^−1
LR	0.25–250/μg mL^−1^a; 0.05–25/μg^b; 250–5000/mg L^−1^c; 20–2000/ng mL^−1^d; 9.4–2400/mg L^−1^e; 5–70/μg mL^−1^f	1–400/μg L^−1
%RSD	<25^a; <6.2^b; <4.5^c; <12.1^e; <6.5^f	<4.2
%Recovery	>75^a; >92^b; >96^c; >43^f	>91.4

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Conclusions

A single drop liquid–liquid-liquid microextraction method coupled with RP-HPLC-UV was developed for the determination of hippuric acid in human urine and serum samples. The method is simple, fast, sensitive, and accurate. The results from validation indicate the proposed method can be applied for routine determination of hippuric acid in biological fluid samples.

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