For the last decade, significant attention has been paid to the occurrence, bioaccumulation and fate of drugs in effluent hospital water. Therefore, the aim of this study was to develop and validate analytical method to identify and quantify the antibiotics tetracycline HCl, (Tetra) doxycycline, (Doxy), ampicillin trihydrate (Ampi), amoxicillin trihydrate (Amoxi) and cephalexin monohydrate (Cefalex). The LCMS instrument used was equipped with with C18 column, (150 mm length x 4.6 mm inner diameter x5 um particle size). The mobile phase was acetonitrile/formic acid (1%) under gradient elution mode. The MS employs ESI unit and quadrupole mass analyzer. The analysis time was less than 15 min. The method was validated in terms of linearity, precision, accuracy, robustness, limit of detection and limit of quantitation, specificity, stability and excellent results were obtained.
Keywords: Development; Optimization; LCMS; Antibiotics; Aquatic environment
Antibiotics are natural, semisynthetic or synthetic drugs used as antibacterial, antifungal or antiparasitic. Antibiotics can be grouped by either their chemical structure or mechanism of action [1-3]. Antibiotics represent a major source of micro pollutants as they may as chemical mixtures that exhibit a wide range of mechanisms of action [4-9,1]. Moreover, they can undergo chemical and/or physical reactions leading different metabolites by the action of microorganisms, as well as by other physical or chemical means, resulting in mixtures with higher toxicities and risks to human health than those of the individual compounds [4,1,10-16]. In contrast to their therapeutic outcome, these antibiotics often disadvantageous for those target and non-target organisms. In addition to this, improperly disposal of unused antibiotics and nonmetabolized antibiotics excreted by humans can all enter the sewer system in low concentrations. However, the use of antibiotics is growing and their input to the aquatic environment is increasing making them of increasing environmental relevance. The increased awareness that synthetic drugs can lead to serious side effects in the environment has prompted researchers to launch several monitoring studies into the most commonly administered compounds in urban wastewater [17-24]. In this work, we developed and validated ESI. LC-MS method for the determination of some antibiotics in hospital waste water.
Antibiotics reference standards tetracycline HCl, (Tetra) doxycycline, (Doxy), ampicillin trihydrate (Ampi), amoxicillin trihydrate (Amoxi) and cephalexin monohydrate (Cefalex) were kindly donated by Azal Pharmaceutical, Company, Khartoum Sudan.
Chemicals and reagents
Acetonitrile, formic acid (HPLC grade), methanol 99% (analytical grade and HPLC grade), phosphoric acid (98%), acetone 99% triethylamine (analytical reagent grade) were purchased from Scharlu, Spain.
Three samples were collected from wastewater (sewerage system) and from different locations in Khartoum North Hospital. The samples were preserved and stored in 500 ml amber borosilicate glass bottles to prevent photo degradation. The samples collected were mixed before cleanup.
Samples pretreatment and clean up
The samples were filtered through 0.45 um filter paper, acidified to pH 3.0 by adding phosphoric (0.1 M) and then were passed through activated C18 cartridge which was activated with 5 ml methanol/water 50:50 (v/v). The cartridge was washed further with 5 ml of acidified water (pH 3.) and then was eluted with 5 ml of triethylamine (5% v/v) in methanol. The eluted solution was evaporated at normal room temperature (28°C). Finally, sample was made to 1 ml by adding water/ acetonitrile 95:5 (v/v) and introduced to the LC-MS instrument where 10 uL were injected.
LC-MS 2020 (Shimadzu Corporation, Kyoto, Japan) equipped with C18 column, (150 mm length x 4.6 mm inner diameter x5 um particle size). Pump mode binary gradient (LC-20AD), flow rate 0.5000 ml/min (Tables 1 and 2).
|Time In min||Module||command||Component A value (formic acid1%) Pump A||Component A value (acetonitrile) Pump B|
|0.01||pump||Pump B conc.||0.00||100|
|20.00||pump||Pump B conc.||70.00||30|
|22.00||pump||Pump B conc.||0||100|
|25.00||pump||Pump B conc.||0||100|
Table 1: Gradient elution programme.
|Auto sampler model||SIL-20AC|
|Enable auto sampler||Use|
|Sample rack||Rack 1.5 ml 105 vials|
|Rinsing volume||500 ul|
|Needle stroke||52 mm|
|Control vial needle stroke||52 mm|
|Rinsing speed||35 ul/s|
|Sampling speed||15 ul/s|
|Purge time||25.0 min|
|Rinse Dip time||0 sec|
Table 2: Auto sampler settings.
The LCMS experimental parameters are shown in Table 3.
|Start time||0.00 min|
|End time||25.10 min|
|Acquisition mode||Scan & SIM|
|Event time||1.00 sec|
|Detector voltage||+1.20 kV|
|Scan speed||938 u/s|
|Nebulizing gas flow||1.5 L/min|
Table 3: Common MS settings.
Preparation of standard solution stock
A weight of exactly 0.05 g of each antibiotic tetracycline HCl, (Tetra) doxycycline, (Doxy), ampicillin trihydrate (Ampi), amoxicillin trihydrate (Amoxi) and cephalexin monohydrate (Cefalex) was transferred into a 50 ml volumetric flask and the volume was completed by the diluents which is acetonitrile and formic acid (1%), (1:1 v/v) and then ultrasonicated. 1 ml of this solution was transferred into a 10 ml flask and was completed to volume by using the same diluent. From this solution, 1 ml was pipettted into a 10 ml flask and was completed to volume by addition of the same diluent as above to obtain a solution of a concentration of 1 ppm.
During the last two decades LCMS has been extensively used in the environmental research for identification and quantification of pollutants and this due to its performance characteristics such as accuracy reproducibility, low detection limit and sensitivity. The current study reports a novel and validated method for quantitative analysis of nine antibiotics commonly found in hospital effluents using LC-MS. Sample preparation and clean up was achieved by using solid-phase technique as it is a powerful sample clean up method in various antibiotic matrices [25-27].
Several gradient programs were tried to achieve the optimum separation of the entire antibiotics standard. Gradient elution was necessary to avoid excessive retention. Well resolved peaks were obtained within short analysis time.
The positive and the negative electrospray ionization (ESI) scan modes were investigated for attaining the highest sensitivity during the method development process. The full scan of the antibiotics mixture in positive mode showed that the signal-to-noise ratios obtained in this mode were higher than those of the in negative mode. Hence, positive mode was used to obtain the precursor ion [M+H] for the qualitative and quantitative analysis [9,17,28-31]. During the method development, the quadrupole mass analyzers operated in selected ion monitoring (SIM) mode where it monitors only a few mass-tocharge ratios. By using electrospray ionization and subsequent analysis produced the chromatogram shown in Figure 1.
Although all peaks were well resolved in this study, LC MS capability allows analysis of co-eluted analytes. This allows fast analysis time and minimal sample preparation. Table 4 shows the precursor ion and the retention time.
Table 4: The ion peaks (M/z) and the retention times of the antibiotic standards.
Method performances and validation
Developing and validation of a method for LC-MS involves demonstrating all the performance characteristics such as linearity, precision, accuracy, limits of detection and quantitation, solution stability and robustness . The linearity of a test procedure is its ability (within a given range) to produce results that are directly proportional to the concentration of analyte in the sample. Acceptability of linearity data is often judged by examining the correlation coefficient (r2) and y-intercept of the linear regression line for the response versus concentration plot. Regression line equations are shown in Table 5. Excellent correlation between the instrumental response and the concentration were obtained.
|Drug||Regression line equation||R2|
Table 5: The linearity testing results.
The precision of the method (intraday) was examined by repeatedly injecting the antibiotic solutions. Precision criteria for an assay method are that the instrument precision and the intra-assay precision (RSD) will be ≤ 2%. 0.28% RSD. The intraday precision was in the range of 1.450-44% (Table 6).
Table 6: The results of repeatability testing.
Excellent values were obtained for interday precision and the values range was 0.78-1.39 (Table 7).
|Day 1||Day 2||Day 3|
Table 7: The results of reproducibility testing.
The accuracy of the method was evaluated by determination of the recovery of the antibiotics at four concentration levels (80,100 and 130%). The accuracy of the method was determined by calculating recoveries of each standard. The results showed good recoveries (Table 8).
Table 8: The accuracy test results.
The limit of detection (LOD) and the limit of quantitation (LOQ) and for the analyzed samples were calculated using the standard deviation of the response (σ) and the slopes (s) i.e. LOD=3.3σ/s and LOQ=10 σ/s. Low detection and quantitation limits were obtained (Table 9).
Table 9: The results of Limit of detection and quantitation testing.
Specificity which is the ability of the method to accurately measure the analyte response in the presence of all potential sample components. The obtained results blank injection showed absence of any interferents. Solution stability of the antibiotics standards solution was also assessed after 6 h room temperature storage. For solutions to be considered stable, the results of the percentage difference between the mean response for the fresh and stored solutions should be ≤ 5.0% (Figures 2a and 2b and Table 10).
|Drug||injection1||injection2||AVG area||std area||std con||found con||AVG RE %|
Table 10: The solution stability test results.
Robustness which is the reliability of an analysis with respect to deliberate variations in method parameters of an analytical procedure should show the reliability of an analysis with respect to deliberate variations in method parameters. The evaluation of robustness should be considered during the development phase. The standard pH was 3.0 and the room temperature was 28°C. These two parameters were varied in order to evaluate the robustness of the methods and excellent results are shown in Table 11.
Table 11: The results of robustness testing.
The developed method was applied for the determination of antibiotics in hospital wastewater samples. The results of the sample analyses are tetracycline HCl, doxycycline, and cephalexin monohydrate with concentrations of 0.124, 0.134 and 0.084 ppm, respectively. Ampicillin trihydrate and amoxicillin trihydrate were not detected (Figure 3).
Such findings necessitate the need for more efficient wastewater treatment plants and stricter quality control measures. However, there numerous routes by which the disposed of antibiotics and other drugs can reach aquatic environment. However, antibiotics persist and degrade slowly, pass through water treatment plants and thereafter transported to sediment or aquatic environment.
Chemical degradation includes hydrolysis oxidation, decarboxylation, isomerization and elimination. Hydrolysis spitting by water, is a potential degradation pathway for organic pollutants in the aquatic environment and it is probably the most commonly encountered mode of drug degradation. Examples of antibiotics that undergo hydrolysis include lactones, amide sand macrolides. The pH has a profound effect in hydrolysis reaction. For instance, at neutral pH, hydrolysis of sulphonamides is very slow whereas lactams hydrolyse under acidic conditions [33-37]. It is noteworthy that the environmental occurrence, persistence, fate and bioaccumulation ability of antibiotics differ depending on their chemical properties and on the environmental conditions [38,39].
A novel LC-MS method was developed for analysis of amoxicillin trihydrate, ampicillin trihydrate, cephalexin monohydrate, norfloxacin HCl, ciprofloxacin, tetracycline HCl, azithromycin, doxycycline and clarythromycin. The method proved to be accurate, precise linear, reproducible and robust. The method can be used conveniently for identification and quantification of these antibiotics in aqueous samples.