Rapid determination of sulfonamides in milk using micellar electrokinetic chromatography with fluorescence detection

Analytica Chimica Acta 552 (2005) 110–115

Rapid determination of sulfonamides in milk using micellar electrokinetic chromatography with fluorescence detection Sushma Lamba, Sunil Kumar Sanghi ∗ , Amit Asthana, Manjusha Shelke Regional Research Laboratory, CSIR, Microfluidics and MEMS, Near Habibganj Naka, Hoshangabad Road, Bhopal, MP 462026, India Received 8 April 2005; received in revised form 18 May 2005; accepted 19 May 2005 Available online 3 October 2005

Abstract A new method was developed for the determination of sulfonamides in milk by using micellar electrokinetic chromatography coupled with fluorescence detection. Separation of fluorescamine-derivatized sulfonamides was accomplished by using a buffer 13.32 mM disodium hydrogen phosphate, 6.67 mM potassium dihydrogen phosphate and 40 mM sodium dodecyl sulphate at pH 7.5 in addition to positive power supply at 21 kV at 25 ◦ C. Detection was performed using UG-11 excitation filter and 495 nm emission filters. The proposed capillary electrophoresis method allows the separation of five sulfonamides within 7 min with a limit of detection of 1.59–7.68 nmol/L for all the sulfonamides considered for present study. A simple sample preparation method with fairly good recoveries 85–114% is also presented in current paper. Inter-day and intra-day validation of the separation method shows fairly good results. Robustness of the method has also been studied. © 2005 Elsevier B.V. All rights reserved. Keywords: Sulfonamides; Fluorescamine; Micellar electrokinetic chromatography; Milk

1. Introduction Sulfonamide group of drugs is often used in veterinary practice for therapeutic and prophylactic purposes [1]. They are also used in the treatment of human infections, but to a lesser extent [2]. Improper administration of these antibiotics can leave residues in edible animal products like meat, milk, egg and fish [3–5]. One of the drugs, sulfamethazine is suspected to be carcinogenic and produce thyroid tumors in rodent [6] and others are known to cause allergic reactions in human. Owing to their potential impact on human health, the European Union has adopted a maximum residue level (MRL) of 100 ␮g/kg in edible animal tissue and 10 ␮g/L in milk [7]. Therefore there is a need for the development of

Abbreviations: MEKC, micellar electrokinetic chromatography; CE, capillary electrophoresis; MS, mass spectrometry; MS/MS, tandem mass spectrometry; FT-IR, Fourier transform infrared; S.D., standard deviation; R.S.D., relative standard deviation ∗ Corresponding author. Tel.: +91 755 2489402; fax: +91 755 2488323. E-mail address: [email protected] (S.K. Sanghi). 0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.05.084

sensitive and selective method for monitoring their residue level in edible animal products. There are several analytical methods for the determination of sulfonamides, among them high performance liquid chromatography with ultra-violet detection (HPLC–UV) is the most widely applied [3,8]. HPLC with photodiode array detection has also been applied for the determination of sulfonamides in milk [9,10]; but the matrix influence from tissue sample reduces the selectivity of the HPLC-UV detection. Therefore either a good matrix cleanup procedure is required [11] or a very selective detector is needed. Gas chromatography coupled with mass spectrometry (GC–MS) methods are relatively sensitive and selective [12,13], but routine residue analysis by these methods are not feasible because of many purification steps required prior to the analysis of thermally labile and non-volatile sulfonamides. However, several methods involving mass spectrometry (MS) for detection such as liquid chromatography mass spectrometry (LC–MS) [14–16], LC tandem mass spectrometry (LC–MS/MS) [17], gas chromatography mass spectrometry, HPLC atmospheric pressure chemical ionization mass

S. Lamba et al. / Analytica Chimica Acta 552 (2005) 110–115

spectrometry (APCI-MS) [18], packed column supercritical fluid chromatography (pSFC) APCI-MS [19], have been successfully used for the determination of sulfonamides. Packed column supercritical fluid chromatography interfaced to FTIR spectrometry has also been applied to determine eight sulfonamides [20], but FT-IR detection is non-specific for such compounds. Capillary zone electrophoresis interfaced with nano-electrospray MS/MS/MS has been successfully applied for the detection of sulfonamides in milk. However, the separation suffers from interferences from salt and fat from milk [21]. Fluorescence spectroscopic methods coupled with LC have been widely applied to the analytical problems, requiring highly sensitive detection. The sensitivity and selectivity of fluorometry after pre- and post-column derivatization have encouraged their widespread use in the analysis of sulfonamides. Post-column derivatization with fluorescamine has been applied for the HPLC determination of sulfonamides in human saliva [22] and in salmon [23,24]. Another approach is the pre-column derivatization of sulfonamides with fluorescamine [25–27] and OPA [28]. HPLC in combination with fluorescence detection have an excellent limit of detection but at the cost of using toxic solvents for separation and prolonged analysis time. Capillary electrophoresis has been proven to be a highly efficient and rapid analytical technique for various applications [29–32]. CE has inherent advantage over HPLC or GC such as highly efficient separations in relatively short time, small sample volumes and almost negligible consumption of organic solvents. CE is not very common for the determination of sulfonamides. Some work has been done on CE in this area, using UV detection [33–35] and amperometric detection [36]. Several others [37–39] have examined the separation of sulfonamides by micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulphate (SDS) as a micellar phase. In the present work, the applicability of capillary electrophoresis (CE) method for the analysis of residual sulfonamides in milk has been evaluated. Present work deals with pre-column derivatization of sulfonamides with fluorescamine followed by MEKC coupled with sensitive fluorescence detection.

2. Materials and methods 2.1. Chemicals and solutions Sulfanilamide, sulfathiazole, sulfamethoxazole, sulfaguanidine, sulfadiazine and sodium acetate were obtained from Sigma (St. Louis, MO, USA). Glacial acetic acid, disodium hydrogen phosphate and potassium dihydrogen phosphate were obtained from E. Merck (India). Sodium dodecyl sulphate (SDS) was used as surfactant and was obtained from BDH (India). Methanol (HPLC grade), hydrochloric acid (AR), acetone (HPLC grade) was from Ranbaxy (India).

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Stock solutions of sulfonamides (10−2 M) were prepared by dissolving appropriate amounts of the sulfonamides in 3 mL of 3 M HCl, and then made up to 10 mL with distilled water. An intermediate composite standard solution and working standard solution were prepared by taking an aliquot of the stock solution and diluting the mixture with distilled water. All solutions were stored in dark at ∼4 ◦ C and were stable for at least 1 month. Fluorescamine {4-phenylspiro[furan-(3H),1-phthalan]-3,3 dione} was obtained from fluka (Buchs, Switzerland) as derivatizing reagent. Fluorescamine (10 mM solution in acetone) was prepared daily and refrigerated when not in use. All other pure analytical quality chemicals were obtained from standard suppliers and used as received. Acetate buffer (pH 5) used for reaction was prepared by mixing 5.01 mM acetic acid and 9.99 mM sodium acetate. The buffer used for the separation of sulfonamides was of pH 7.5 and was prepared by mixing 13.32 mM disodium hydrogen phosphate, 6.67 mM potassium dihydrogen phosphate and 40 mM sodium dodecyl sulphate (SDS). 2.2. Instrumentation and separation conditions A Prince-C 255 instrument with programmable injector and high voltage source (Prince Technologies, The Netherlands) was used for the experiments. Separation was carried out at 21 kV applied voltage (during reaction optimization 20 kV was applied). Fused silica capillary with internal diameter of 75 ␮m was purchased from Composite Metal Services Ltd. (Worcestershire, UK). A capillary of 53.2 cm total length was used as separation column. A 1 cm detection window was created by burning off the coating at 30.2 cm from the capillary inlet. For the study of reaction pH, a 54.4 cm capillary was used. Samples were introduced hydrodynamically, by applying pressure of 40 mbar for 12 s. For fluorescence detection, an ARGOS 250 B instrument (Flux Instruments, Switzerland) equipped with a 75 W xenon–mercury lamp was used. The excitation light was filtered through a Schott glass UG-11 filter and a 495 nm cut off filter was applied for the limited light. For the decoupling of emitted light glycerol was applied between the capillary and optical cone. The voltage used for PMT (photo multiplier tube) was 800 V. For data processing, DAx 7.1 Data Acquisition and analysis Software (Prince Technologies, The Netherlands) was used. 2.3. Electrophoretic method Before first use, a new capillary was charged by rinsing with 0.1 M NaOH for 40 min, followed by a 15 min rinse with deionized water. At the beginning of each day the capillary was regenerated by rinsing with methanol for 10 min, followed by water for 5 min, 1 M hydrochloric acid for 10 min, water for 5 min, 0.1 M sodium hydroxide for 20 min, water for

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5 min and background electrolyte (BGE) for 20 min. Before each run the capillary was first rinsed with 0.1 M NaOH for 2 min. At the end of each day the capillary was flushed with deionized water for 10 min followed by drying with air for 2 min. 2.4. Sample preparation The buffalo’s milk used in this experiment was purchased from the local market. For the extraction of sulfonamide from milk, 100 ␮L of 0.09 M hydrochloric acid was added to 1 mL portion of the sample and diluted to 10 mL with methanol. After shaking for 1 min the sample was allowed to stand for 15 min and the supernatant was derivatized for final analysis. 2.5. Derivatization procedure To 1 mL of sample/working standard in acetate buffer (pH 5), 40 ␮L of fluorescamine solution (10 mM) was added and stirred vigorously for 1 min. The sample was allowed to stand at room temperature for 20 min before injection.

Fig. 1. Effect of pH on fluorescence yield. Capillary: 54.4 cm (30.2 cm effective length) × 75 ␮m I.D. BGE: 13.32 mM disodium hydrogen phosphate, 6.67 mM potassium dihydrogen phosphate (pH 7.5) and 40 mM sodium dodecyl sulphate (SDS). Applied voltage: 21.4 kV. Peaks: 1, sulfanilamide; 2, sulfaguanidine; 3, sulfadiazine; 4, sulfamethoxazole; 5, sulfathiazole.

3. Result and discussion

several minutes to several hours, as also observed by Stein et al. [41], compared to milliseconds in aqueous medium. Under optimized reaction condition the reaction completes in 20 min and does not further increase with reaction time (Fig. 1).

3.1. Optimization of the derivatization conditions

3.2. Optimization of separation conditions

3.1.1. Effect of pH on reaction It was found that maximum yield was obtained when the selected sulfonamides were derivatized in an aqueous solution of pH between 3 and 5. At higher pH the yield decreases because of probable hydrolysis of fluorescamine and at lower pH the yield decreases due to protonation of amino group of sulfonamides [40]. The best combination of pH and buffer concentration was found in acetate buffer pH 5 (15 mM), which was selected for further studies.

3.2.1. Effect of pH The changes in pH were found to affect the migration behavior of sulfonamides (Fig. 2). At pH 7.5 (13.32 mM disodium hydrogen phosphate and 6.67 mM potassium dihydrogen phosphate) the effective mobilities of sulfadiazine, sulfamethoxazole, sulfathiazole changes effectively, however baseline separation of the pair sulfanilamide and sulfaguanidine was not observed. Hence at pH 7.5 sodium dodecyl sulphate (SDS) was added for micellar electrokinetic separation of the sulfonamides.

3.1.2. Effect of amount of fluorescamine and co-solvent The concentration of fluorescamine is another factor, which affect the reaction yield [40]. The fluorescence yield was optimized by using different amount of 10 mM fluorescamine while keeping the sulfonamide concentration constant. It appeared that 40 ␮L of 100 mM fluorescamine, corresponding to 0.4 mM concentration is sufficient to achieve the maximum yield. At higher reagent amounts in the reaction mixture, the yield decreases due to fluorescence quenching by one of the hydrolysis product of fluorescamine [40]. It was observed that reaction mixture becomes turbid when higher concentration of reagent (>0.5 mM) was added in the reaction mixture. It was found that the reagent concentration needed to achieve maximum fluorescence also depended upon the amount of organic co-solvent. On increasing the percentage of acetone on the reaction mixture a relative decrease in fluorescence intensity was observed. It slows down the reaction of sulfonamides with fluorescamine from

Fig. 2. Effective mobility of sulfonamides obtained at varied pH (between 7.0 and 9.0). Operating conditions and peak numbering as in Fig. 1.

S. Lamba et al. / Analytica Chimica Acta 552 (2005) 110–115

3.2.2. Effect of SDS concentration The pH studies make it clear that a more powerful separation technique like MEKC is needed to separate the five sulfonamides. In current studies SDS the most common surfactant used in MEKC has been employed for the separation of sulfonamides. Background electrolyte (BGE) system containing 20 mM phosphate buffer (pH 7.5) at different SDS concentration up to 50 mmol/L was used to study the effect of SDS concentration on resolution. The results obtained are shown in Fig. 3, where effective mobilities were plotted against SDS concentration. An increase in migration time and resolution of the five fluorescamine-derivatized sulfonamides was observed when the SDS concentration in the BGE increased. The results indicate markable change in retention behavior of sulfathiazole and sulfaguanidine. Retention of both the compound increases with increase in SDS concentration. This is in accordance with the typical partition behavior of the compounds. Baseline resolution of all the fluorescamine-derivatized drugs was obtained at SDS concentrations ≥40 mmol/L. The migration and resolution of fluorescamine derivatives of sulfonamides are significantly affected by the SDS concentration. Sodium dodecyl sulphate (40 mM) at pH 7.5 was found to be optimum for the separation of all the five fluorescamine-derivatized sulfonamides. Further increase in SDS concentration increases separation time as well as current. Separation of all five fluorescaminederivatized sulfonamides is depicted in Fig. 4. The plate number under the optimum conditions was in the range of 22 000–105 000. 3.2.3. Effect of injection time Effect of increase in the volume of the sample injection on resolution was also studied. Hydrodynamic injection of 40 mbar for 12 s (81.07 nL) was found to be the best compromise between good limits of detection and resolution. Increase in injection volume lead to better limit of detections but at the cost of resolution.

Fig. 3. Effect of SDS concentration of the BGE on the effective mobilities of the fluorescamine derivatives. Other operating conditions and peak numbering as in Fig. 1.

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Fig. 4. Separation of a standard mixture of five derivatized sulfonamides by MEKC. Applied voltage: 21 kV. Other operating conditions are as for Fig. 1. Sample concentration: 10−6 mol/L of each compound. Peaks: *, electro-osmotic flow; 1, sulfanilamide; 2, sulfaguanidine; 3, sulfadiazine; 4, sulfamethoxazole; 5, sulfathiazole.

3.3. Validation of the method 3.3.1. Recovery studies The extraction recovery was determined by comparing the corrected peak areas of sulfonamides extracted from spiked milk samples with that of the unextracted standards containing the same amount of sulfonamides. For the determination of sulfonamides in milk three spiked samples at concentrations of 5, 10 and 15 ␮mol/L were used. The three spiking standards were prepared by transferring milk (1 mL) into three different 10 mL calibrated flasks, then adding the spiked solution to the flasks and diluting with methanol to 10 mL to give the desired concentrations [19]. For the sample preparation and derivatization same procedure is applied as described in Sections 2.4 and 2.5. The results are shown in Table 1. The average recovery ranged from 85 to 114% in the concentration range of 5–15 ␮mol/L. The reproducibility of the extraction procedure is determined by three replicates at fortification level 10 ␮mol/L. Each replicate represents the mean of three values. The relative standard deviation of recoveries were less than 4% for the sulfonamides, except for sulfathiazole (9.75%). 3.3.2. Precision The precision of the analytical method is determined by comparing the effective mobilities of sulfonamide standards. Table 2 represents inter- and intra-day repeatability in terms of percentage R.S.D. in effective mobilities and corrected peak area. Inter-day repeatability was measured within 15 days. As indicated in Table 3 excellent intraday (%R.S.D. < 0.83) and inter-day (%R.S.D. < 1.77) reproducibility for effective mobilities were achieved. Percentage R.S.D. for corrected peak area was also fairly good for both intra-day (%R.S.D. < 4.74) and inter-day (%R.S.D. < 4.38) except for sulfathiazole.

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114 Table 1 Recovery at different spiking level Sulfonamides

Recovery (%) 5 ␮mol/L

10 ␮mol/L

Average ± S.D. Sulfanilamide Sulfaguanidine Sulfadiazine Sulfamethoxazole Sulfathiazole

87.33 85.12 114.63 100.28 94.34

± ± ± ± ±

3.45 2.29 4.62 4.22 9.62

15 ␮mol/L

%R.S.D.

Average ± S.D.

3.96 2.69 4.03 4.20 10.20

98.80 96.69 101.22 101.87 105.61

± ± ± ± ±

1.10 1.60 1.12 0.75 4.91

%R.S.D.

Average ± S.D.

1.12 1.65 1.11 0.74 4.66

101.15 97.40 98.90 100.84 108.40

± ± ± ± ±

%R.S.D.

2.51 4.60 2.45 0.52 3.13

2.49 4.73 2.48 0.52 2.89

Table 2 Precision of analytical method Sulfonamides

Sulfanilamide Sulfaguanidine Sulfadiazine Sulfamethoxazole Sulfathiazole a b c

%R.S.D. corrected peak area

%R.S.D. effective mobility

Intra-dayb

Inter-dayc

Intra-day

Inter-day

1.30 2.54 1.30 1.05 4.74

1.87 1.92 1.87 4.36 12.17

0.83 0.75 0.69 0.72 0.68

1.65 1.28 1.77 1.74 1.30

Sulfadiazine was taken as internal standard. n = 6. n = 6 and each value is average of three replicates.

3.3.3. Calibration curve and sensitivity Calibration curves were plotted between the concentration range 50–1000 nmol/L and were found to be linear over this range. The correlation coefficients, r, of the standard curves, concentration limit of detection (LOD) and limits of quantitation (LOQ) for five sulfonamides under the selected conditions are summarized in Table 3. Limit of detection is defined as concentration, which gives a signal to noise ratio of 3:1. The detection limits is between the range 1.59–7.68 nmol/L, which was far below the maximum residue level for milk. The LOQ is defined as average of background plus 10 standard deviations. The electropherogram of the blank milk sample (Fig. 5B) shows a small peak having same effective mobility as those of sulfaguanidine in the spiked milk sample (Fig. 5A), which denotes to 2.02 ␮mol/L sulfaguanidine in the milk. Sulfadiazine was used as internal standard.

Fig. 5. The electopherogram of milk samples (A) spiked with five sulfonamides at 10 ␮mol/L. (B) Blank milk sample with internal standard sulfadiazine at 10 ␮mol/L. Peaks: *, electro-osmotic flow; 1, sulfanilamide; 2, sulfaguanidine; **, unknown; 3, sulfadiazine; 4, sulfamethoxazole; 5, sulfathiazole. Other operating conditions as in Fig. 4.

3.3.4. Robustness Robustness relates to the capacity of the method to remain unaffected by small but deliberate variations introduced in

Table 3 Calibration curve and detection limits Sulfonamides

y = a + bxa a ± S.D.

Sulfanilamide Sulfaguanidine Sulfadiazine Sulfamethoxazole Sulfathiazole a b c

1.18 × 10−5

−7.6 × 10−5 3.17 × 10−6 1.54 × 10−5 4.47 × 10−5

Sa ± ± ± ± ±

1.18 × 10−5 7.55 × 10−5 3.17 × 10−5 1.54 × 10−5 4.47 × 10−5

b

1.12 × 10−5 1.98 × 10−5 1.3 × 10−5 5 × 10−6 2.62 × 10−5

b ± S.D.

Sb

4827.65 ± 4827.65 3935.59 ± 3935.59 3449.12 ± 3449.12 1350.35 ± 350.35 1340.82 ± 1340.82

21.78 38.43 25.18 9.69 50.84

x: concentration (mol/L); y: peak area/migration time; a: intercept; b: slope. Standard error in intercept. Standard error in slope.

c

LOD (nmol/L)

LOQ (nmol/L)

1.59 2.36 3.29 7.68 7.04

5.3 7.88 10.97 25.59 23.47

r2 0.9999 0.9996 0.9997 0.9997 0.9942

S. Lamba et al. / Analytica Chimica Acta 552 (2005) 110–115 Table 4 Robustness of the method Parameter changed

NaOH rinsing (1.5, 2.0, 2.5 min) Buffer rinsing (1.5, 2.0, 2.5 min) Injection time (11.4, 12.0, 12.6 s) SDS concentration (36, 40, 44 mM) Phosphate concentration (18, 20, 22 mM) Separation temperature (23, 25, 27 ◦ C)

Parameter studied Effective mobilities (%R.S.D.)

Corrected peak area (%R.S.D.)

0.72–1.0 1.0–9.6 – 1.7–2.9 5.5–6.2 0.8–1.6

– – 3.6–14.2 − − −

method parameters. The most relevant factors to investigate are the electrolyte composition, injected volume, separation temperature and rinse time etc. These factors are varied around the value set in the method to reflect the changes likely to arise in different test environment. The results of robustness study are summarized in Table 4 indicating that the method is fairly robust under different test conditions.

[7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]

4. Conclusion

[24]

An MEKC method has been developed for the determination of sulfonamides in milk after pre-column derivatization with fluorescamine. The method has been validated and the results showed acceptable performances. The LOD and LOQ for all sulfonamides were adequate for practical analysis in milk sample. The simplified extraction procedure, including extraction with methanol, enables quantitative determination of five of the most used sulfonamides at concentration far below maximum residue level (10 ␮g/L) The advantage of MEKC for sulfonamides analysis is the elimination of the need for expensive organic solvents and column. Analysis by CE is also simple, rapid and robust.

[25] [26] [27]

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