Native fluorescent detection with sequential injection chromatography for doping control analysis
© Idris and Alnajjar; licensee Chemistry Central Ltd. 2013
Received: 16 April 2013
Accepted: 5 July 2013
Published: 28 August 2013
Sequential injection chromatography (SIC) is a young, ten years old, separation technique. It was proposed with the benefits of reagent-saving, rapid analysis, system miniaturization and simplicity. SIC with UV detection has proven to be efficient mostly for pharmaceutical analysis. In the current study, a stand-alone multi-wavelength fluorescence (FL) detector was coupled to an SIC system. The hyphenation was exploited for developing an SIC-FL method for the separation and quantification of amiloride (AML) and furosemide (FSM) in human urine and tablet formulation.
AML and FSM were detected using excitation maxima at 380 and 270 nm, respectively, and emission maxima at 413 and 470 nm, respectively. The separation was accomplished in less than 2.0 min into a C18 monolithic column (50 × 4.6 nm) with a mobile phase containing 25 mmol/L phosphate buffer (pH 4.0): acetonitrile: (35:65, v/v). The detection limits were found to be 12 and 470 ng/mL for AML and FSM, respectively.
The proposed SIC-FL method features satisfactory sensitivity for AML and FSM in urine samples for the minimum required performance limits recommended by the World Anti-Doping Agency, besides a downscaled consumption of reagents and high rapidity for industrial-scale analysis of pharmaceutical preparations.
KeywordsSequential injection chromatography High performance liquid chromatography Amiloride Furosemide Method validation
The association of AML and FSM furnishes a valuable natriuretic agent with a diminished kaliuretic effect and minimizes the risk of alkalosis in the treatment of refractory oedema associated with hepatic cirrhosis or congestive heart failure . Due to the benefits of their simultaneous use, AML and FSM are being prepared as binary dosage forms. Accordingly, the development of assay methods for those two drugs is desirable for the purpose of quality control. In this issue, various analytical techniques were exploited including high performance liquid chromatography [4, 6, 7], spectrophotometry [8–10], fluorometry  and electroanalytical .
On the other hand, athletes use diuretics, in general, for flushing previously taken prohibited substances with forced diuresis  to achieve acute weight loss. Hence, the World Anti-Doping Agency (WADA) prohibits the use of diuretics . Besides being an ethically condemned practice, the risk to the athletes’ health has to be considered since they are generally self-administered in a wrong manner; i.e. overdoses, interactions with other drugs or even the use of drugs of illicit origin [14–16]. Evidently, a sensitive and reliable analytical method to determine diuretics in urine and/or plasma is a prerequisite in sport activities. Toward this end, WADA establishes a minimum detection capability for testing methods called the Minimum Required Performance Limits (MRPL). This is to ensure that all doping control laboratories can report the presence of prohibited substances uniformly. The limit for each analyte in the class of diuretics is 250 ng/mL [17, 18].
The dominant techniques used for screening diuretics in control urinalysis are GC and HPLC. However, both techniques have the limitations of the high cost of instrumentation and maintenance. Moreover, other challenges in GC namely are the low volatility of the compounds and the necessity of the additional step of derivatization. HPLC has also the limitation of large consumption of solvent volumes, which is due to the continuous flow of mobile phase and large instrumentation dimension.
Recently, sequential injection chromatography (SIC) was introduced to overcome some challenges in separation techniques . In principle, the procedure of SIC is based on a sequential injection, i.e. a discontinuous-flow approach, of a mobile phase and samples. The separation process is usually carried out into a monolith column using programmable miniaturized modules. The association of the three approaches of the discontinuous-flow approach, monolith separation column and system miniaturization renders SIC procedure simple, rapid and reagent-saving [20–22]. On the other side, the major limitation of SIC is the limited pressure of the syringe pump. The maximum is 900 psi. This causes back-pressure in separation column and hence limits the use of long separation column and hence reduces the separation capability of many analytes. However, nineteen chromatographic peaks for amino acids were observed in an SIC profile in a previous study . Another limitation of SIC is that the limited volume of the syringe. The commercially available syringe volume is 10 mL, which might not be sufficient for eluting all compounds from a separation column. However, this problem could be solved by refilling the syringe.
In the current study, a SIC system was coupled with a fluorescence (FL) detector to provide an analytical method for the separation and quantification of AML and FSM in human urine and pharmaceutical formulation. The capabilities of that couple were exploited in terms of reagent-saving, analysis time, sensitivity and selectivity.
Results and discussion
A short monolith column (50 × 4.6 mm) was examined and, initially, it has been found to be sufficient for the separation of the two drugs. With respect to column dimension, the practicable flow rate of 20–40 μL/s in SIC was tested [24–30]. It is well known that high flow rate accelerates analysis and sharpens peaks. In contrast, high flow rates increases the back-pressure in a separation column. Accordingly, the optimum flow rate set in the current study was 25 μL/s. On the other hand, with respect to peak height and peak shape, the practicable range of sample volume is 40–60 μL [24–30]. At a large sample volume, peak height was significantly improved while acceptable peak shape was not achieved. Hence, the optimum sample volume has been found to be 30 μL.
Minimum and maximum levels of pH, buffer concentration and percentage volume of acetonitrile adopted for the 2 3 full-factorial design matrix for method optimization
Buffer concentration (mmol/L)
Percentage volume of acetonitrile (%)
2 3 full-factorial design matrix a and further experiments b for screening the effect of pH buffer concentration and percentage volume of acetonitrile on resolution (R), retention time (t R , min) and peak area
Optimal analytical conditions of the SIC-FL method
(50 × 4.6 mm)
Mobile phase composition
25 mm phosphate : acetonitrile (35:65 v/v, pH 4.0)
Sample volume (μL)
Flow rate (μL/s)
Excitation wavelength (nm)
Emission wavelength (nm)
The validation metrics including linearity, recovery and simple and complex precision as well as the limits of detection and quantitation were determined from measurements.
For linearity studies, samples were prepared using the highest calibrators, namely 100 μg/mL AML and 200 μg/mL FSM. Serial dilutions were made to achieve AML concentrations of: 0.1, 0.5, 1.0, 2.0, 3.0 and 4.0 μg/mL; and FSM concentrations of: 2.0, 3.0, 4.0, 6.0, 8.0 and 10.0 μg/mL. Linearity was determined via the least squares linear regression analysis of the data obtained from the average of three replicates from each of the levels described above. A previous spectrofluorimetric method  reported linear ranges of 3.7 × 10-4-0.8 and 1.2 × 10-3-4.0 μg/mL for AML and FSM, respectively. Despite these ranges are lower than the corresponding of the current SIC-FL method, the latter are sufficient for drug detection in urine and more suitable for pharmaceutical analysis.
Validation metrics results of the SIC-FL method
Consumed mobile phase volume (mL)
Total analysis time (min)
Sample frequency (samples/h)
Retention time (min)
PAa = 1.6387Cb + 566.28
PAa = 26.667Cb + 23.333
Linear range (μg/mL)
Within-run precision (RSD, %)
Between-run precision (RSD, %)
Recovery in urine samples (%)
Recovery in tablets (%)
99.1 for 1.0 μg/mL
98.7 for 8.0 μg/mL
97.5 for 0.5 μg/mL
98.1 for 4.0 μg/mL
97.0 for 0.5 μg/mL
96.5 for 0.5 μg/mL
The LOD was determined at a signal-to-noise ratio of 3 whereas the LOQ was determined at a signal-to-noise ratio of 10. The noise was assessed using drug-free human urine samples after SPE. Interestingly, the LOQs of AML and FSM (Table 4) achieved from the current SIC-FL match the MRPL that was recommended by WADA [17, 18]. A previous HPLC screening test was presented for some diuretics of doping interest in human urine . In that method, the LOD for AML and FSM were 0.750 and 0.125 μg/mL, respectively. These levels are not in consistence with the current SIC-FL method. This could be attributed to the use of liquid-liquid extraction for sample treatment and UV for the detection in the previous HPLC method  while SPE and fluorescence detection were used in the current SIC method.
The proposed SIC-FL method was applied to four urine samples. All samples were subjected to SPE. One sample was free from AML and FSM in order to examine the efficiency of SPE for sample clean-up and to calculate the LODs and LOQs. The chromatogram of one sample as an example is depicted in Figure 2a. Acceptable baseline was obtained indicating acceptable efficiency of the sample clean-up step in the adopted SPE procedure . The other samples were spiked with different quantities of AML and FSM as described in the subsection entitled “Method validation”. As an example, Figure 2b shows the chromatogram of 3.0 μg/mL AML and FSM in a urine sample. The pre-concentration factor obtained from SPE was 5. Hence, the level of AML and FSM injected into the SIC-FL system was 0.6 μg/mL. The recovery obtained (Table 4) was acceptable indicating the accuracy of the SPE procedure  and the proposed SIC-FL method.
Unique SIC analytical features
One of the interesting advantages of the proposed SIC-FL method over HPLC methods is mainly the reagent-consumption. The total volume of the consumed reagent, i.e. mobile phase for column conditioning and separation, was 4.5 mL. Hence, in routine analysis, SIC consumes milliters per day versus centiliters per day for HPLC. As previously mentioned, the reduction of reagent consumption in SIC is due to the discontinuous-flow, downscaled-dimension of instrumentation and the use of monolithic column. Accordingly, the waste production of SIC is less than that of HPLC and hence the frontal is greener than the latter. On the other side, the use of monolithic column and the miniaturization of SIC work hand in hand to provide a rapid analysis. The total analysis time including column conditioning and elution, without SPE, was 4.7 min. Hence, the sample frequency was 13 samples/h. Furthermore, the instrumentation simplicity of SIC offers less instrumentation cost. In addition, the simplicity in SIC instrumentation makes its maintenance cost less.
The detector was 2475 Multi λ Florescence Detector from Waters (Milford, CT, US). The light source was a xenon lamb with excitation wavelength in the range of 200–900 nm. The detector was equipped with excitation and emission monochromators.
The solid phase extraction (SPE) columns of 6-mL size, which were used for urine sample treatment, were Discovery DSC-18. They were supplied by Supleco (Bellefonte, PA, US).
Chemicals, reagents and samples
Double-distilled deionized water was used throughout the experimental work. All chemicals and reagents were of analytical reagent grade. AML hydrochloride hydrate, FSM, acetonitrile, sodium hydrogen phosphate, ortho-phosphoric acid were purchased from Sigma-Aldrich (Taufkirchen, Germany).
Preparation of reagents and samples
100 μg/mL of AML and 200 μg/mL of FSM as stock standard solutions were prepared by dissolving appropriate amounts in mobile phase. Working standard solutions were prepared by dilution in an appropriate way.
10 mL of urine samples were obtained from eight healthy volunteers. All samples were filtered through Whatman® paper No 1. Two samples were then subjected directly to SPE without spiking drugs. The other samples were spiked in duplicate with three concentrations of each drug as described in the subsection entitled “Method validation”. Thereafter, the three replicate samples were subjected to SPE. 2.5 mL of urine were cleaned through the SPE columns by 3 mL of water and eluted with 2 mL of methanol. The methanolic extract was evaporated to dryness under nitrogen stream and reconstituted in 2 mL mobile phase. Blank urine was treated in same manner .
Frumil® tablets (5 mg of AML hydrochloride and 40 mg of FSM), which were prepared by Sanofi-Aventis (Dublin, Ireland), were examined. Twenty tablets were accurately weighed and finely powdered. Three portions were accurately weighed and transferred into 100-mL calibrated flasks. The first portion was equivalent to 5 mg of AML and 40 mg of FSM. The second portion was equivalent to 2.5 mg of AML and 20 mg of FSM. The third portion was equivalent to 1.25 mg of AML and 10 mg of FSM. The drugs were extracted by the mobile phase with shaking and filtration. The solutions were diluted fifty-times. The recovery in tablet formulation was examined using a previous HPLC method as a reference .
A rapid protocol controlling the proposed SIC procedure was programmed. 1.0 mL of the mobile phase was aspirated through the check valve in the syringe pump at a flow rate of 150 μL/s. For column conditioning, the mobile phase was introduced into the separation column through port-2 and the guard column (Figure 4) at a flow rate of 30 μL/s. The syringe was filled again with 3500 μL of the mobile phase at a flow rate of 150 μL/s. 30 μL of standards/samples were loaded into the holding coil through port-3 to port-10 at a flow rate of 10 μL/s. The sample and mobile phase were then injected into the guard and separation columns through port-2 at a flow rate of 25 μL/s. During this step, the fluorescence detector was set at excitation and emission wavelengths as presented in Table 3.
A fluorescence detector was hyphenated to an SIC system to generate a sensitive and direct method for the separation and quantification of AML and FSM. The hyphenation also permitted a simple, inexpensive, rapid and reagent-saving procedure. The SIC-FL method was validated and it demonstrated to be reliable for the determination of both drugs, being linear, accurate and precise. Therefore, the SIC-FL method can be considered suitable for the quantification of both drugs in human urine samples. The SIC-FL method is also thought to be ideally suited for a rapid routine analysis for the quality control of pharmaceutical products.
Dr. Abubakr M. Idris is a Sudanese associate professor of analytical chemistry at the Department of Chemistry, College of Science, King Khalid University, Abha, Saudi Arabia. Idris has authored more than sixty-five papers and books published in international refereed journals and conferences. His research focuses on developing microfluidic analytical technologies and their methodologies. He has some publications on environmental issues as well. Idris joints the editorial board of American Journal of Analytical Chemistry, Development in Analytical Chemistry and Novus Scientia Journals. He also gained awards for funding more than fifteen research projects from various institutes. On the other hand, Idris has many activities on the issues of academic development and quality. He is currently the chairman of the Unit of Academic Development and Quality, College of Science, King Khalid University.
This work was supported by the grant from King Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia, award number M-T-5-8. The financial contribution is gratefully acknowledged. Dr. Idris thanks the Department of Chemistry, College of Science, King Khalid University for encouraging him to join the KACST Research Team at. Dr. Alnajjar also thanks the Department of Chemistry, College of Science, King Faisal University for allowing them to conduct this research in their laboratories.
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