Journal of Chromatography A, 1360 (2014) 305–311

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Simple semi-automated portable capillary electrophoresis instrument with contactless conductivity detection for the determination of ␤-agonists in pharmaceutical and pig-feed samples Thi Anh Huong Nguyen a , Thi Ngoc Mai Pham a , Thi Tuoi Doan a , Thi Thao Ta a , Jorge Sáiz c , Thi Quynh Hoa Nguyen a , Peter C. Hauser b,∗∗ , Thanh Duc Mai b,d,∗ a Department of Analytical Chemistry, Faculty of Chemistry, Hanoi University of Science, Vietnam National University, Hanoi - 19 Le Thanh Tong, Hanoi, Viet Nam b University of Basel, Department of Chemistry, Spitalstrasse 51, 4056 Basel, Switzerland c Department of Analytical Chemistry, Physical Chemistry and Chemical Engeneering, University of Alcalá, Ctra, Madrid-Barcelona km 33.6, Alcalá de Henares, Madrid, Spain d Centre for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Nguyen Trai Street 334, Hanoi, Viet Nam

a r t i c l e

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Article history: Received 30 May 2014 Received in revised form 18 July 2014 Accepted 24 July 2014 Available online 1 August 2014 Keywords: Semi-automated portable instrument Capacitively coupled contactless conductivity detection (C4 D) Capillary electrophoresis (CE) ␤-Agonists

a b s t r a c t An inexpensive, robust and easy to use portable capillary electrophoresis instrument with miniaturized high-voltage capacitively coupled contactless conductivity detection was developed. The system utilizes pneumatic operation to manipulate the solutions for all flushing steps. The different operations, i.e. capillary flushing, interface rinsing, and electrophoretic separation, are easily activated by turning an electronic switch. To allow the analysis of samples with limited available volume, and to render the construction less complicated compared to a computer-controlled counterpart, sample injection is carried out hydrodynamically directly from the sample vial into the capillary by manual syphoning. The system is a well performing solution where the financial means for the highly expensive commercial instruments are not available and where the in-house construction of a sophisticated automated instrument is not possible due to limited mechanical and electronic workshop facilities and software programming expertise. For demonstration, the system was employed successfully for the determination of some ␤-agonists, namely salbutamol, metoprolol and ractopamine down to 0.7 ppm in pharmaceutical and pig-feed sample matrices in Vietnam. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Portable instrumentation has gained much interest thanks to its advantages of rapid availability of results, elimination of complications with sample storage and transport, and lower cost than conventional bench-top analytical systems. Capillary electrophoresis (CE), with its advantageous properties of high separation efficiency, short analysis time, low consumption of chemicals, ease of installation, operation and maintenance, is a particularly good candidate for portable analytical instrumentation. Since its first

∗ Corresponding author at: Centre for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Nguyen Trai Street 334, Hanoi, Viet Nam. Tel.: +33 651 37 79 49. ∗∗ Corresponding author. Tel.: +41 61 267 1003; fax: +41 61 267 1013. E-mail addresses: [email protected] (P.C. Hauser), [email protected], [email protected] (T.D. Mai). http://dx.doi.org/10.1016/j.chroma.2014.07.074 0021-9673/© 2014 Elsevier B.V. All rights reserved.

appearance in 1998 in a custom-built format [1], and in 2001 in a commercial version [2,3], portable CE (P-CE) has been developed in different configurations. Detailed discussions of P-CE up to 2013 can be found in two recent reviews [4,5]. A more recent design of P-CE has been developed by our group based on a flowcell interface and pneumatic operation [6]. A modified version of this P-CE was then successfully applied for determination of nitrogen mustard degradation products in water [7] and scopolamine in forensic evidence [8]. In most of the cases, capacitively coupled contactless conductivity detection (C4 D) was the detection method of choice for P-CE. Low power consumption, the possibility for miniaturization, high versatility and ease in construction and operation are among the notable positive features of this detector. For fundamental aspects of C4 D, see for example [9–16] whereas recent applications of CE-C4 D can be found in several reviews [17–21]. It is relatively simple to construct a P-CE system based on fully manual operation [22] and good results have, for example, been obtained for environmental studies carried out in Siberia even in

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winter time [23,24]. On the other hand, to carry out all procedures by hand can be challenging, especially when working in the field. Furthermore, in contrast to optical detection, C4 D allows the employment of relatively narrow capillaries which have the important advantage of giving better separation efficiencies [25–28]. In particular the manual flushing of capillaries with internal diameters of 50 ␮m or less is difficult because of the relatively high required pressures. This has been carried out with syringes, which need to be coupled to the capillary with pressure tight fittings and pressed hard by hand for extended periods. For this reason automated instruments have been developed in our laboratories recently [6–8]. On the other hand, this feature significantly complicates the construction of the instruments and imparts the need for a certain level of expertise in mechanical construction, electronic design and computer programming. The assembly of such a system with limited workshop facilities is thus not possible. The instrument described herein represents a compromise between the level of automation and ease of construction and operation. A semi-automated P-CE system, relying on pneumatic operation for capillary/interface flushing and on siphoning for hydrodynamic sample injection was built. The different steps of operation are activated with a multi-position rotary switch without requiring a computer-based control program. Basing system control on a simple switch made its construction possible with limited expertise and workshop infrastructure. While computer control via software is a more elegant solution, it would require the design and construction of a purpose made electronic interface and programming expertise. To reduce the size and weight of the P-CE system, a different high-voltage module from those in our previous portable instruments was employed, which is available at significantly lower cost while still maintaining the important high voltage and current monitoring feature. A further significant advantage of the instrument described herein compared to the automated instruments recently described by us is the fact that the sample volume can be considerably smaller than what is needed for the automated injectors of those instruments [6–8]. The new robust system was demonstrated for the separation of some ␤-agonists, namely salbutamol, ractopamine and metoprolol in different pharmaceutical and pig-feed sample matrices in Vietnam. These compounds have been illegally used for pig feed to promote reduction in body fat and enhance growth in swine. Pig meat contaminated with ␤-agonists once consumed can lead to intoxication in humans (see [29] and references listed therein). Meanwhile, several ␤-agonists, especially salbutamol, metoprolol have been widely used as drugs to treat pulmonary diseases in humans as well as in cattle and animals. To the best of our knowledge, CE-C4 D has previously been used only for determination of salbutamol in syrups [30], and there has been no study using this technique on separations of ␤-agonist mixtures in various samples so far.

2. Experimental 2.1. Chemicals and materials Arginine (pKa 9.09), phosphoric acid, ascorbic acid and acetic acid were of analytical or reagent grade and purchased from Fluka (Buchs, Switzerland) or Merck (Darmstadt, Germany). Salbutamol, metoprolol (+)- tartrate and ractopamine hydrochloride were purchased from Sigma–Aldrich (Saint-Quentin Fallavier, France) and stored at 4 ◦ C before use. They were allowed to reach ambient temperature prior to analysis. Polyimide coated fused silica capillaries of 50 ␮m I.D. and 365 ␮m O.D. (from Polymicro, Phoenix, AZ, USA) with total (L) and effective (Leff ) lengths of 60 cm and 53 cm respectively were used for separations. Before use, the capillary was preconditioned

with 1 M NaOH for 10 min and deionized water for 10 min prior to flushing with buffer. Deionized water purified using a system from Water Pro (Labconco, Kansas City, MO, USA) was used for the preparation of all solutions and for sample dilution if required. For sample preparation, a pharmaceutical syrup containing salbutamol sulfate obtained from Solmux Broncho (United International Pharma, Binh Duong, Vietnam) was diluted with deionized water (15 g/50 mL). Pharmaceutical tablets (5 mg, from Dorpharma, Hanoi and Plendil Plus, AstraZeneca AB, Sodertalje, Sweden) were pulverized and then dissolved with 5 mL deionized water (for salbutamol-containing tablets) or 5 mL MeOH (for metoprolol containing tablets). The obtained solutions were ultrasonicated for 10 min and filtered through 0.45 ␮m membranes. For metoprolol tablets, a centrifugation step at 8000 rpm for 10 min was required to precipitate the suspension-creating excipients before filtration. Further dilution with deionized water was then carried out if needed. Pig feed samples (2 g) were pulverized, 10 mL MeOH/H2 O mixture (v/v ratio = 4/1) added and then vortex mixed. The obtained suspensions were ultrasonicated for 2 h and then filtered through 0.45 ␮m membranes before injection into the CE system. Note that these sample preparation steps cannot be automated and therefore require manual operations, but are simple and reliable and therefore could be carried out by a person with little experience. For cross check, samples were also analyzed with HPLC–MS at the National Institute for Food Control (NIFC), Hanoi, Vietnam, according to a method reported elsewhere [31,32]. 2.2. Instrumentation The fluidic part is based on a flow-cell interface machined in a Plexiglas block (3 cm × 2 cm × 2 cm) (for more details see [6,33]) and two solenoid 2-port valves with 30 psi holding pressure purchased from NResearch (116T021, Guemligen, Switzerland). All fluidic connections were made with 0.02 in. I.D. and 1/16 in. O.D. Teflon tubing and with polyether ether ketone flangeless nuts and ¼-28 ferrules purchased from Upchurch Scientific (Oak Harbor, WA, USA). Pneumatic pressurization was achieved with a standard cylinder of compressed air, or with an air pump and a reservoir for field measurements, whose outlet pressure was adjusted to 1 bar with a regulator. The electrophoresis section is based on a miniature and inexpensive Spellman high voltage (HV) unit (UM20*4, 12 V 200 ␮A, Pulborough, UK) to provide a maximum 20 kV of pre-selected polarity. The HV end of the capillary was isolated with a safety cage made from Perspex, which was equipped with a microswitch to interrupt the HV on opening. The rotary electronic switch (Art. No. RA01C04BSFMSOX, 3 poles, 4 positions) was purchased from Farnell (Zug, Switzerland). Detection was carried out with a miniaturized HV – C4 D built in-house according to the design reported previously [34,35]. A function generator in the surface mount technology (SMT) format (XR2206, Exar, Fremont, California, USA) was used to obtain the sine wave excitation voltage of 20 Vp-p at 400 kHz, which was then boosted to 200 Vp-p using a purpose-built transformer. The pick-up amplifier (OPA602, Texas Instruments, Austin, TX, USA), the rectifier (AD630, Analog Devices, Norwood, MA, USA) as well as the low-noise operational amplifiers acting as a low-pass filter (OPA2227, Burr-Brown product from Texas Instrument, Austin, Texas, USA) were also in the SMT configuration. The resulting signals were recorded with an ADC-20 data acquisition system (Pico Technology, St. Neots, UK) connected to the USB-port of a personal computer. For powering the electrophoretic and fluidic parts, a lithium battery pack of 14.8 V and a capacity of 6.6 Ah ¨ (CGR 18650CG 4S3P, Contrel, Hunenberg, Switzerland) fitted with a voltage regulator for production of a 12 V output was used. A separate pair of smaller Li-ion batteries with a capacity of 2.8 Ah each (CGR 18659CG 4S1P, Contrel), which was fitted with positive

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and negative 12 V regulators, provided the split ±12 V supply for the C4 D circuitry. Alternatively, mains power can be utilized when available.

3. Results and discussion 3.1. System design and operation A diagram of the fluidic connections of the system is shown in Fig. 1. Fluidic propulsion through the flow-cell interface which hosts the ground electrode and one end of the capillary was carried out by pressurization of the background electrolyte reservoir. Electronic on/off control of the two solenoid valves situated before and after the flowcell interface (designated as V1 and V2, respectively) allows divergence of the electrolyte into either the capillary for capillary flushing or to waste for interface rinsing. Note that a somewhat similar pneumatically actuated flushing device for capillary electrophoresis had been reported by Kaljurand et al. [36]. Hydrodynamic injection of the sample was implemented by lifting the HV end of the capillary to a certain height during a pre-determined duration at the closing of V1 and opening of V2. This siphoning action was chosen for the injection step for two reasons: (1) to minimize the sample amount required for each injection (in the upper nL-range, rather than the at least 20 ␮L sample volume which is required for each injection with our automated portable systems [6–8]), and (2) to simplify the system design which in turns renders the construction much more facile than the computer-controlled versions and is thus possible with modest workshop facilities. Note that the detector should be positioned close to the ground-end of the capillary to facilitate the movement of the HV-end during injection. A photograph detailing the arrangement of the instrument is given in Fig. 2. For the first time to the best of our knowledge, a miniaturized Spellman HV module with dimensions of 120 mm (L) × 38 mm (W) × 25 mm (H) and a weight of 200 g was employed for the construction of a purpose-built CE system. Its advantages featured over other previously reported HV modules (see more details in [37]) are the compact size with low power consumption which is suitable for P-CE and the possibility of direct monitoring of the current and voltage during electrophoresis with digital displays. For mechanical rigidity, all components of the system were integrated into a Perspex case with the dimensions of 40 cm (w) × 28 cm (d) × 21 cm (h) and a weight of only 6 kg. The fluidic parts were

Fig. 2. Photograph of the instrument. (1) Safety cage for application of HV, (2) Electronic board for control of HV on/off and magnitude, which accommodates the HV adjustment trimmer and two digital screens for the monitoring of voltage and current values, (3) miniaturized HV – conductively coupled contactless conductivity detector, (4) flowcell interface housing one capillary end and the ground electrode, (5) rotary selector switch, (6) Box containing solenoid valves, (7) gas pressurized buffer container.

arranged in the front for facile operation. On the left was the safety Perspex cage which contains the HV electrode dipped in a buffer vial and the capillary was introduced from the top. The solenoid valves, the flowcell interface and the rotary selector switch were mounted on a separated Perspex box and were arranged on the right together with the detector. The electronic components were positioned at the back and the electrical controls of the system are schematically illustrated in Fig. 3. The HV magnitude could be adjusted by turning a trimmer situated on the top of the Perspex case and the current and voltage values could be monitored with two separated digital screens positioned next to the trimmer (denoted as the potentiometer and ammeter in Fig. 3). The rotary selector switch, which is composed of two synchronized 4-position selector ones, was used to control simultaneously the two solenoid valves (denoted as Valve 1 and Valve 2 in Fig. 3). By turning the rotary selector switch to one of the four pre-defined positions, either of the desired operations including capillary flushing, interface rinsing, siphoning injection and electrophoretic separation was activated. While not quite as convenient as software control it still greatly improves the handling of operations and is a very robust solution.

Separation capillary

Sample Regulating Valve

Pressure Gauge

Pt C4D

2-Port Valve

GND V2

V1

Flowcell interface

2-Port Valve Waste

Pt HV

Buffer Safety cage

Compressed air

Pressurized Buffer Container

Fig. 1. Diagram of the fluidic connections of the instrument. Pt denotes the two platinum electrodes for application of the HV for separation. The flow-cell interface is grounded at all times.

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Safety microswitch

On/Off indicator LED

HV indicator LED

On/Off +12V Power supply

A

B

1 2 3 4 1 2 3 4

kV µA V

50mV

A

Spellman HV UM20*4 HV setting

Valve 1

A)

B)

+5V C)

Valve 2

Sal Met Rac

Rotary switch

300

Fig. 3. Schematic drawing of the electrical controls of the instrument. HV: high voltage.

An overview of a typical sequence of the operations is given in Table 1. The protocol starts with the rinsing of the interface with electrolyte on the opening of both V1 and V2. Then the capillary is flushed by pushing the solution into the interface while closing V2. Hydrodynamic injection is then carried out by lifting the HV end of the capillary dipped in the sample vial to a certain height for a determined duration while V2 remains open and V1 is closed. This is the only step for which manipulation of the capillary by hand is needed. After switching back the HV end of the capillary to the buffer vial located inside the safety cage, electrophoretic separation is triggered by activating the HV at the closing of V1 and opening of V2. The electrode in the interface remains grounded at all times. 3.2. CE-C4 D determination of ˇ-agonists in different sample matrices The control of ␤-agonists contents in pig-feed and pharmaceutical samples in Vietnam has been so far implemented using HPLC–MS methods. Chromatographic methods have been the most popular method for determination of ␤-agonists (see for example [31,32] [38–41]). In these methods, samples must be transported into the laboratory and stored for a certain period of time. For economic reason, the analyses are carried out only when there are a sufficient number of collected samples. These methods therefore cannot be used for quick and inexpensive evaluation of the content(s) of ␤-agonist(s) in a random sample and on site. These are requirements when the presence of illegally used ␤-agonists in suspected pig-feed needs to be verified. Herein, we propose a new, simple and inexpensive method based on CE-C4 D which is suitable for mobile deployment to determine some ␤-agonists, namely salbutamol, metoprolol and ractopamine in different sample matrices, using the developed portable semi-automated CE system. Note that while CE has been used for such purpose [29,42,43], the employment of C4 D as the detection technique has been so

350

400

450

500

550

600

Migration time (s) Fig. 4. Electropherograms for the separation of salbutamol (Sal), metoprolol (Met) and ractopamine (Rac) (60 ppm) with different buffer compositions. (A) Arginine 10 mM adjusted to pH 4.9 with phosphoric acid, (B) arginine 10 mM adjusted to pH 4.9 with ascorbic acid, (C) arginine 10 mM adjusted to pH 4.9 with acetic acid. CE conditions: hydrodynamic injection 10 cm for 20 s; voltage: +18 kV; capillary: fused-silica, 50 ␮m I.D., Lt = 60 cm (Leff = 53 cm).

far communicated only once for determination of salbutamol in syrups [30]. 3.2.1. Optimization of the CE-C4 D conditions for separation of selected ˇ-agonists Since the pKa values of the amine groups of salbutamol, metoprolol and ractopamine are 9.3, 9.7 and 9.4 respectively, these compounds should be separated as positively charged ions under acidic or nearly neutral CE conditions. From our preliminary tests with different pH ranging from 4 to 6, using buffers composed of arginine and acetic acid of different concentrations, the best signal to noise ratios were achieved at pH 4.9 (data not shown). Note that a pH higher than 6 was not used in order to avoid the presence of an elevated electroosmotic flow. Accordingly, different buffer compositions at pH 4.9 were tested, using arginine (10 mM) adjusted with phosphoric, ascorbic or acetic acid. The effect of these buffer compositions on separation performance in terms of peak heights and resolutions are shown in Fig. 4. As can be seen, the best separation performance was obtained with a buffer containing arginine and acetic acid. The most stable baseline was recorded as well in this case, which most likely is related to the conductivity of the buffer. Note that the negative-going peaks are due to a reduction of the conductivity when the analytes passed the detector. Buffer concentration is another parameter that needs to be optimized, as according Mai and Hauser [26], capillaries of different internal diameters require different buffer concentrations to give the best signal-to-noise ratios with CE-C4 D. An effort was therefore undertaken to establish the optimal concentration of arginine and acetic acid for the separation of salbutamol, metoprolol and ractopamine using capillaries of 50 ␮m ID. The concentrations of

Table 1 Typical operation protocol of the semi-automated P-CE system. Step

Operation

Position of the rotary selector switch

Time (s)

V1

V2

HV

1 2 3 4

Flushing of the flow-cell interface Flushing of the capillary Sample siphoning injection Electrophoretic separation

Interface flushing Capillary flushing Injection Separation

3 120 5 Variable

Open Open Closed Closed

Open Closed Open Open

Off Off Off On

Remark

Manual siphoning

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20 mV

309

Matrix cations

50mV

Matrix cations Sample 3

Rac

A)

Sample 2

Sal

B)

Sample 1

Sal

Sal 200

250

300

350

400

450

0

200

400

600

800

Migration time (s)

500

Migration time (s) Fig. 5. Electropherograms for determination of salbutamol in syrup. (A) Without and (B) with standard addition of 40 ppm salbutamol. Electrolyte: arginine 10 mM adjusted to pH 4.9 with acetic acid. Other conditions as in Fig. 4.

arginine were varied from 5 mM to 20 mM and the pH was always maintained at 4.9 using acetic acid. It was experimentally found that an increase in arginine concentration from 10 mM to 20 mM led to a decrease in peak height (data not shown). On the other hand, arginine concentrations lower than 10 mM did not lead to a further signal enhancement. Accordingly, a buffer of 10 mM arginine adjusted to pH 4.9 with acetic acid was employed for separation of salbutamol, metoprolol and ractopamine. The performance data for this buffer is shown in Table 2. The detection limits achieved for the conditions are in the order of 0.5 ppm and calibration curves were acquired up to 150 ppm. For higher concentrations peak overlapping would occur. The reproducibilities of the measurements of peak areas and migration times were found to be up to 6% and 1% respectively. 3.2.2. Applications The separation of salbutamol in a pharmaceutical syrup sample using the CE-C4 D optimized conditions can be seen in Fig. 5. The presence of matrix cations in the sample did not adversely affect the determination of this ␤-agonist as their arrivals at the detection point occurred before that of salbutamol. A shortcoming of CE is the possible drifts in migration time when using uncoated fused silica capillaries, which can occur due to modification of the surface when injecting different solutions and resulting changes in the electroosmotic flow. This can be exacerbated for a simple non-thermostated instrument used in environments with changing temperatures. For the analysis of the samples, the fluctuation of migration times was therefore found to be more pronounced than the tests with the

Fig. 6. Determination of salbutamol and ractopamine in three pig-feed samples with CE-C4 D. Electrolyte: arginine 10 mM adjusted to pH 4.9 with acetic acid. Other conditions as in Fig. 4.

standard solution reported in Table 2. In cases when the peak identity was not clear, confirmation was carried out by spiking with a standard, which is not a serious complication. For the determination of the ␤-agonist concentrations, the standard addition method was employed. In Table 3 the concentrations of salbutamol and metoprolol determined in different samples with the CE-C4 D and HPLC–MS methods are given. Good agreement between the results obtained from two methods (3–13%), as well as between the experimental data and those provided by the manufacturers (12–28%) was achieved. The CE-C4 D method was then used to determine salbutamol and ractopamine in 3 pig-feed samples. The presence of these ␤-agonists can be visualized as shown in the electropherograms in Fig. 6. Cross-checked results using HPLC–MS are given in Table 4. Good agreement was found where deviations of the results obtained with CE-C4 D from those obtained with HPLC–MS (reference values) were less than 13%. Note that such deviations can be expected due to possible analytical bias between two completely different methods. The good statistical correspondence between the two methods can additionally be demonstrated with the Student t-values (see Tables 3 and 4) where the calculated t-values (tcal ) are always smaller than the critical value of Student’s tdistribution for a 99% confidence interval (tref (P=99 , f=2) = 9.9). To evaluate the sample recovery for the sample preparation procedure, ‘clean’ pig-feed samples were spiked with these ␤-agonists of different concentrations (i.e. 50 ppm and 100 ppm) and determined with CE-C4 D. The recoveries for salbutamol, metoprolol and ractopamine were found to be 99.2%, 103.7% and 98.8%, respectively. Although no officially referenced values of ␤-agonist contents could

Table 2 Calibration ranges, detection limits (LODs) and reproducibility for the determination of ␤-agonists with the P-CE system. Conditions: Electrolyte solution: arginine 10 mM adjusted to pH 4.9 with acetic acid. Sample injection: hydrodynamic, 10 cm for 20 s. Voltage: +18 kV. Capillary: fused-silica, 50 ␮m id, Lt = 60 cm (Leff = 53 cm). Analyte

Range (ppm)a

Correlation efficient r2

LODb (ppm)

RSD % MTc (n = 6)

RSD % PAd (n = 6)

Salbutamol Metoprolol Ractopamine

1.7–150 2.3–150 2.3–150

0.9993 0.9991 0.9997

0.5 0.7 0.7

0.8 0.8 0.9

6.2 4.1 4.7

a b c d

5 concentrations. Based on peak heights corresponding to 3 times the baseline noise. Migration time, RSD was measured with standard concentrations of 40 ppm. Peak area, RSD was measured with standard concentrations of 40 ppm.

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Table 3 Concentrations of salbutamol, metoprolol in pharmaceutical samples determined with CE-C4 D and HPLC–MS. Drugs

Concentration obtained with CE-C4D (␮g/g) (n = 3)

Concentration obtained with HPLC–MS (␮g/g)

Concentration stated by the manufacturer (␮g/g)

tcal b

Salbutamol in Solmux Broncho syrup Salbutamol tablet Metoprolol in Plendil Plus tablet

0.17 ± 0.01a 3.5 ± 0.2 64 ± 1

0.15 3.4 60

0.20 4.0 50

3.5 0.9 6.9

a This concentration (␮g salbutamol in 1 g syrup) was calculated from the result obtained after dilution with deionized water (15 g syrup in 50 mL) and measurement with CE-C4 D. |x−| √ b The Student t-values (tcal ) were calculated according to the following equation: tcal = S n where x and  are the mean result measured with CE-C4 D and the result measured with HPLC–MS, respectively. S is the standard deviation and n is the number of replicates.

Table 4 Concentrations of salbutamol, ractopamine in pig-feed samples determined with CE-C4 D and HPLC–MS. Samples

Concentration obtained with CE-C4 D (mg/kg) (n = 3)

Pig feed 1 – containing salbutamol Pig feed 2 – containing salbutamol Pig feed 3 – containing ractopamine

297 ± 9 71 ± 2 36 ± 1

a

Concentration obtained with HPLC–MS (mg/kg) (reference value) 281 82 31

Deviation from the reference value (%) 5.7 −13.4 13.9

tcal a 3.1 9.5 8.7

tcal values were calculated according to the equation as in Table 3.

be obtained as they are used illegally, as a rule of thumb in Vietnam their contents of 20–500 ppm were added into pig feeds just 15–30 days before slaughter. Due to this short period, it is imperative to seize the suspected samples at the right moment and the determination of the ␤-agonist contents should be carried out immediately without sample storage so that the decision of confiscation of the suspected material can be made as quickly as possible. It is expected that the use of the developed CE-C4 D method with the portable semi-automated system could open the floor to quick and efficient control of the contents of ␤-agonists illegally used in pig-feed samples. 4. Conclusions A portable semi-automated CE system using pneumatic operation was constructed and demonstrated for application to the determination of ␤-agonists in different sample matrices. The instrument is inexpensive, simple in construction and can therefore be assembled with little effort. The system control via a rotary selector switch rather than a computer-based program, rendered the construction possible with little expertise and allows significant reduction of construction cost and complexity, while still maintaining the automatic feature in almost all steps in the protocol. The system is therefore a well performing solution where the financial means for the highly expensive commercial instruments are not available and/or the workshop facilities are modest. The approach taken was found to be very robust in day to day operation. A further advantage of the instrument compared to recent automated versions described by us is the possibility of using much smaller sample volumes. Using the developed system, an analytical procedure was developed for CE-C4 D determination of salbutamol, metoprolol and ractopamine in pharmaceutical and pig-feed samples. Mobile deployment of the system for on-site applications is envisaged, but it may of course also be employed as a low cost instrument in the laboratory. Acknowledgements The authors are grateful for partial financial support by the Swiss National Science Foundation (grant 200020-137676). We would like to thank Dr. Le Thi Hong Hao and MSc. Vu Thi Trang at the National Institute for Food Control (Vietnam) for providing the pigfeed samples and HPLC–MS operation.

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