Research Article - (2018) Volume 9, Issue 2

ESI-LC/MS Method Development and Validation for the Determination of Some Selected Antibiotics in Hospital Wastewater

Elhag DE1*, Abdallah BS1, Hassan M2,3 and Suliman A1
1Analytical Research Centre, UMST University, Khartoum, Sudan
2Swiss Tropical & Public Health Institute, Socinstrasse 57, 4051 Basel, Switzerland
3Basel University, Peterplatz 1, 4001, Basel, Switzerland
*Corresponding Author: Elhag DE, Analytical Research Centre, UMST University, Khartoum, Sudan, Tel: +249 183 228614 Email:


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.

Materials and Methods

Antibiotics Standards

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
25.10 pump stop 0  

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 mode Before/After
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
Polarity Positive
Event time 1.00 sec
Detector voltage +1.20 kV
Start m/z 100.00
End m/z 1000.00
Scan speed 938 u/s
Interface ESI
DL temperature 250°C
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.

Results and Discussion

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].

LC-MS soptimization

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.


Figure 1: Chromatogram of the antibiotics standard.

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.

Drug Retention time M/z
Tetra 12.034 445.15
Doxy 14.014 445.15
Ampi 10.862 350.1
Amoxi 8.773 366.15
Cefalex 10.858 348.1

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 [32]. 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
tetra Y=86865x-14669 0.999
Doxy Y=1E06x+11923 0.999
Ampi Y=37168x-2454 0.998
Amoxi Y=41663x-1983 0.998
Cefalex 51884x+22985 0.999

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).

Drug 1 2 3 4 5 6 AVG SD RSD
tetra 1756479 1759324 1781687 1759548 1793867 1753518 1767404 16391.91 0.93
Doxy 1685461 1650101 1679713 1621835 1652958 1639810 1654980 24072.5 1.45
Ampi 102656 103065 103160 103330 104035 103103 103224.8 455.1032 0.44
Amoxi 552041 551651 554843 561591 556520 571101 557957.8 7382.376 1.32
Cefalex 1429264 1428162 1431949 1434767 1442267 1425304 1431952 6001.813 0.42

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
tetra 34824.82 1.93428 20111.91 1.083573 14413.97 0.78
Doxy 33701.61 1.917657 32774.64 1.942115 15980.39 0.95
Ampi 1556.515 1.542105 918.1362 0.81053 964.5576 0.855
Amoxi 3936.037 0.642846 10674.47 1.702397 7980.729 1.27
Cefalex 10462.19 0.702811 15534.94 0.994861 12659.74 0.81

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).

    80% 100% 130%
tetra std con 101.9607 99.99998 101.8249
Doxy 1 101.6675 100 101.0471
Ampi 1 99.5806 99.87969 103.0607
Amoxi 1 99.17957 99.71749 104.4347
Cefalex 1 100.0237 99.87456 103.6223

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).

tetra 0.02 0.12
Doxy 0.02 0.18
Ampi 0.02 0.22
Amoxi 0.08 0.8
Cefalex 0.02 0.05

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 %
tetra 1813011 1856073 1834542 1856073 1 0.9884 98.83997
Doxy 1788795 1687575 1738185 1687575 1 1.02999 102.999
Ampi 108434 113276 110855 113276 1 0.978627 97.86274
Amoxi 572373 627026 599699.5 627026 1 0.956419 95.64189
Cefalex 1502397 1561519 1531958 1561519 1 0.981069 98.10691

Table 10: The solution stability test results.


Figure 2a: Antibiotics standard chromatogram.


Figure 2b: Antibiotics standard chromatogram after six hours from the first injection.

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.

Parameter Drug Run1 Run2 Run3 AVG SD RSD
29°C tetra 1761821 1759775 1768001 1763199 4282.631 0.24289
  Doxy 1560235 1558049 1532373 1550219 15493.69 0.999452
  Ampi 99096 99071 101316 99827.67 1288.995 1.29122
  Amoxi 548559 537823 546558 544313.3 5709.144 1.048871
  Cefalex 1382887 1401628 1399124 1394546 10174.6 0.729599
27°C tetra 1787640 1768801 1789080 1781840 11315.32 0.635036
  Doxy 1677628 1649295 1647241 1658055 16982.09 1.024218
  Ampi 98033 98845 98586 98488 414.7758 0.421144
  Amoxi 541732 546526 546459 544905.7 2748.68 0.504432
  Cefalex 1371988 1383966 1367474 1374476 8522.859 0.620081
PH3.1 tetra 1713358 1704663 1714488 1710836 5376.036 0.314234
  Doxy 1571982 1554607 1544523 1557037 13889.89 0.892072
  Ampi 92592 92732 94447 93257 1032.945 1.107632
  Amoxi 491749 497037 503680 497488.7 5978.31 1.201698
  Cefalex 1296192 1316877 1319020 1310696 12606.74 0.961835
PH2.9 tetra 1601163 1615916 1621137 1612739 10359.14 0.642332
  Doxy 1576493 1554135 1556553 1562394 12270.09 0.785339
  Ampi 95074 94744 95123 94980.33 206.1318 0.217026
  Amoxi 501861 511828 513200 508963 6188.651 1.215933
  Cefalex 1312012 1326165 1341340 1326506 14666.97 1.105684

Table 11: The results of robustness testing.

Sample Analysis

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).


Figure 3: chromatograms of the antibiotic found in hospital effluent sample.

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.


Citation: Elhag DE, Abdallah BS, Hassan M, Suliman A (2018) ESI-LC/MS Method Development and Validation for the Determination of Some Selected Antibiotics in Hospital Wastewater. Pharm Anal Acta 9: 578.

Copyright: ©2018 Elhag DE, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.