Validation and application of an immunochromatographic test to detect four macrolides and two lincosamides in raw cow milk.

In this study, an immunochromatographic test (using the Charm QUAD2® Test) was used to screen for residual macrolides and lincosamides in raw cow's milk. The validation parameters (selectivity/specificity, detection capability (CCß), and ruggedness) were in agreement with the requirements of[EC] 2021. The selectivity of the immunochromatographic test was verified by the negative results of microbiological tests. The false-positive rate was 0%. The CCß values of the immunochromatographic test for various antibiotics in milk were as follows: erythromycin 0.02 mg/kg, spiramycin 0.1 mg/kg, tilmicosin 0.025 mg/kg, tylosin 0.05 mg/kg, lincomycin 0.15 mg/kg, and pirlimycin 0.15 mg/kg. The determined CCß values were lower than the respective maximum residue limits (MRLs; regulatory limits in Japan) for milk, except for lincomycin (equal to the MRL). The presence of antibiotic groups other than macrolides and lincosamides did not interfere with the specificity of the test. It showed no significant difference in lot-to-lot repeatability. The results obtained by the two researchers showed no significant differences. Finally, the test was applied to milk samples obtained from a tylosin-treated cow. The outcome was positive and in agreement with the results of the chemical analytical and microbiological methods. Therefore, this validated immunochromatographic test is expected to be suitable for routine analysis to ensure milk safety.

[Historical Antibiotic Stress Changed the Effects of Sulfamethoxazole and Trimethoprim on Activated Sludge: ARGs and Potential Hosts].

The co-exposure of antibiotics has important effects on antibiotic resistance genes (ARGs) and microbial community aggregation in wastewater treatment plants (WWTPs). However, it is unclear whether differences in historical antibiotic exposure stress can determine responses of microbes and ARGs to combined antibiotics. By selecting a high concentration (30 mg·L-1) of sulfamethoxazole (SMX) and trimethoprim (TMP) as historical exposure stress conditions, the effects of SMX and TMP-combined pollution on ARGs, bacterial communities, and their interactions were explored in short-term experiments. Based on high-throughput quantitative PCR, a total of 13 ARGs were detected, and the absolute abundance was 2.21-5.42 copies·µL-1 (logarithm, DNA, the same below). Among them, sul2, ermB, mefA, and tetM-01 were the main subtypes in the samples, and the absolute abundance was between 2.95 and 5.40 copies·µL-1. The combined contamination of SMX and TMP could cause the enrichment of ARGs and mobile genetic elements (MGEs); however, their effects on each subtype were different, and the historical legacy effect of SMX was higher than that of TMP. Under the different exposure histories, the co-occurrence and co-exclusion patterns existed between ARGs. Moreover, MGEs (especially intI-1) were significantly correlated with sulfonamides (sul1 and sul2), tetracyclines[tet(32)], and macrolide-lincosamide-streptogramin (MLSB) resistance genes (ermB). Based on the full-scale classification of microorganisms, it was found that the microbial community structure of various groups responded differently to combined pollution, and the conditionally abundant taxa (CAT) were obviously enriched. Thauera, Pseudoxanthomonas, and Paracoccus were the dominant resistant bacterial genera. Furthermore, a total of 31 potential hosts of ARGs were identified with network analysis, which were dominated with conditionally rare taxa (CRT). Particularly, Candidatus_Alysiosphaera and Fusibacter were positively correlated with most of the ARGs, being the common protentional hosts. Importantly, some rare genera (RT, Variibacter, Aeromonas, Cloacibacterium, etc.) were potential hosts of transposon IS613, which played an important role in the proliferation and spread of ARGs. In conclusion, this study revealed the legacy effects of historical antibiotic stress on ARGs and their hosts, which could provide new ideas and theoretical basis for reducing ARGs pollution in WWTPs.

Analytical Approaches in Official Food Safety Control: An LC-Orbitrap-HRMS Screening Method for the Multiresidue Determination of Antibiotics in Cow, Sheep, and Goat Milk.

The presence of unauthorized substances, such as residues of veterinary medicines or chemical contaminants, in food can represent a possible health concern. For this reason, a complete legislative framework has been established in the European Union (EU), which defines the maximum limits allowed in food and carries out surveillance programs to control the presence of these substances. Official food control laboratories, in order to ensure a high level of consumer protection, must respond to the challenge of improving and harmonizing the performance of the analytical methods used for the analysis of residues of authorized, unauthorized, or prohibited pharmacologically active substances. Laboratories must also consider the state of the art of the analytical methodologies and the performance requirements of current legislation. The aim of this work was to develop a multiresidue method for the determination of antibiotics in milk, compliant with the criteria and procedures established by Commission Implementing Regulation (EU) 2021/808. The method uses an LC-Orbitrap-HRMS for the determination of 57 molecules of antibiotic and active antibacterial substances belonging to different chemical classes (beta-lactams, tetracyclines, sulfonamides, quinolones, pleuromutilins, macrolides, and lincosamides) in bovine, ovine, and goat milk samples. It provides a simple and quick sample pretreatment and a subsequent identification phase of analytes, at concentrations equal to or lower than the maximum residual limit (MRL), in compliance with Commission Regulation (EU) 2010/37. The validation parameters: selectivity, stability, applicability, and detection capability (ccß), are in agreement with the requirements of Commission Implementing Regulation (EU) 2021/808 and demonstrated the effectiveness of the method in detecting veterinary drug residues at the target screening concentration (at the MRL level or below), with a false positive rate of less than 5%. This method represents an effective solution for detecting antibiotics in milk, which can be successfully applied in routine analyses for official food control plans.

Multi-class, multi-residue determination of 132 veterinary drugs in milk by magnetic solid-phase extraction based on magnetic hypercrosslinked polystyrene prior to their determination by high-performance liquid chromatography-tandem mass spectrometry.

A quantitative multi-class multi-residue analytical method was developed for the determination of veterinary drugs in milk by high-performance liquid chromatography - tandem mass spectrometry (HPLC-MS/MS). A total of 132 veterinary drugs investigated belonged to almost 15 classes including sulfonamides, ß-lactams, tetracyclines, quinolones, macrolides, nitrofurans, nitroimidazoles, phenicols, lincosamides, pleuromutilins, macrocyclic lactones, quinoxaline antibiotics, benzimidazoles, anthelmintics, coccidiostats and some others. A magnetic solid-phase extraction procedure was developed using magnetic hypercrosslinked polystyrene (HCP/Fe3O4) for the sample preparation prior to HPLC-MS/MS without deproteinization step. The results indicated recoveries of 85-107% for 14 sulfonamides, 85-120% for 13 ß-lactams, 89-115% for 4 tetracyclines, 82-119% for 14 quinolones, 82-115% for 8 macrolides, 97-109% for 4 nitrofurans, 84-115% for 10 nitroimidazoles, 89-114% for 3 phenicols, 86-111% for 3 lincosamides, 97-102% for 2 pleuromutilins, 72-88% for 4 macrocyclic lactones, 87-104% for 4 quinoxaline antibiotics, 76-119% for 21 benzimidazoles, 79-115% for 12 anthelmintics, 81-118% for 12 coccidiostats and 75-119 % for 5 unclassified drugs, with relative standard deviations (RSDs) of less than 20%, and the LOQs ranged from 0.05 to 1 µg kg-1. This methodology was then applied to field-collected real milk samples and trace levels of some veterinary drugs were detected.

High-throughput method based on a novel thin-film microextraction coating for determining macrolides and lincosamides in honey.

A high-throughput method using a new ZIF-8@GO thin-film microextraction coating was established for determining macrolides and lincosamides in honey. The coating preparation parameters (ZIF-8@GO synthesis conditions, coating material proportions, dipping time) and analysis parameters (sample diluent solvent, adsorption and desorption conditions using the ZIF-8@GO coating) were optimized. The optimized parameters were: diluent solvent sodium carbonate/sodium bicarbonate buffer solution (pH 9), adsorption time 45 min, desorption time 5 min, desorption solvent 45:40:15 v/v/v methanol/acetonitrile/water. The extracted targets were determined by ultra-high performance liquid chromatography tandem mass spectrometry. The recoveries of 10 analytes were 67.5-107.2% and the detection and quantification limits were 0.1-0.4 and 0.4-1.4 µg/kg, respectively. The method could analyze 96 samples per run. The minimal manual time and effort is required since the bulk of the sample processing is fully automated. It was a useful and efficient method for monitoring drug residues and was successfully used to analyze real samples.

Rapid determination of antibiotic residues in cereals by liquid chromatography triple mass spectrometry.

Antibiotics may be present in agricultural soils through the application of organic amendments as fertilizers or by irrigation of fields with recycled water. As a result of these agricultural practices, antibiotics in soil can lead to their uptake by plants, entering in this way the food chain. Studies on the levels of antibiotics in cereal samples are scarce in the available literature. In this work, an analytical method was developed for the determination of 19 antibiotics (fluoroquinolones, sulfonamides, tetracyclines, and lincosamides) in four types of cereal grains (wheat, barley, rice, and oat). Ultrasound-assisted matrix solid-phase dispersion was selected as extraction technique with recoveries of target analytes ranging from 73 to 127% for the four cereals analyzed. Limits of quantification obtained ranged from 0.8 to 5.8 ng g-1. Compared with methods described for the analysis of antibiotics in cereals, the developed method uses a lower volume of extraction solvent and very good recoveries were obtained for all compounds. The validated method was applied to the analysis of different types of cereals samples, harvested from agricultural fields or purchased from local supermarkets. The analysis of the five cereal samples grown in fields with 3 years of consecutive organic amendments revealed that none of the nineteen antibiotics selected were found in any sample. Eleven commercial samples of cereals of different types and presentations were analyzed and enrofloxacin was detected in one rice sample; the presence of enrofloxacin in cereals or its incorporation into crops from soil or water not previously reported. Graphical abstract.

Expression of resistance genes instead of gene abundance are correlated with trace levels of antibiotics in urban surface waters.

In this study, antibiotic resistance to macrolide-lincosamide-streptogramin B (MLSB) antibiotics in total microbial community in surface water in a coastal urban city was measured using a modified fluorescence in situ hybridization (FISH) technique. This FISH technique quantified the rate of antibiotic resistance to MLSB antibiotics through targeting methylation site of A2058 of 23S rRNAs resulting from expressed erythromycin ribosome methylation (erm) genes. Correlations between the rates of MLSB resistance measured by FISH and macrolide concentrations was stronger than that between the relative abundance of erm genes and macrolide concentrations, especially in residential areas where the main detected antibiotics were macrolides. These results suggest that trace levels of antibiotics in environmental waters, which was as low as 40 ng L-1, may still play important roles in the development and spread of antibiotic resistance. Additionally, methylation as a result of erm gene expression, instead of erm gene abundance, was a better indicator of selective pressure of trace level macrolides. The rates of MLSB resistance varied significantly among land use types, suggesting that anthropogenic activities are important factors to select for erm gene expression in the environment. Microbial community analysis of representative surface water samples showed that relatively high rates of MLSB resistance were observed in Alphaproteobacteria (42%), Acidobacteria (36%), Bacteroidaceae (32%), Chloroflexi (27%), and Betaproteobacteria (20.2%).

The analysis of tetracyclines, quinolones, macrolides, lincosamides, pleuromutilins, and sulfonamides in chicken feathers using UHPLC-MS/MS in order to monitor antibiotic use in the poultry sector.

In The Netherlands, all antibiotic treatments should be registered at the farm and in a central database. To enforce correct antibiotic use and registration, and to enforce prudent use of antibiotics, there is a need for methods that are able to detect antibiotic treatments. Ideally, such a method is able to detect antibiotic applications during the entire lifespan of an animal, including treatments administered during the first days of the animals' lives. Monitoring tissue, as is common practice, only provides a limited window of opportunity, as residue levels in tissue soon drop below measurable quantities. The analysis of feathers proves to be a promising tool in this respect. Furthermore, a qualitative confirmatory method was developed for the analyses of six major groups of antibiotics in ground chicken feathers, aiming for a detection limit as low as reasonably possible. The method was validated according to Commission Decision 2002/657/EC. All compounds comply with the criteria and, as a matter of fact, 58% of the compounds could also be quantified according to regulations. Additionally, we demonstrated that a less laborious method, in which whole feathers were analyzed, proved successful in the detection of applied antibiotics. Most compounds could be detected at levels of 2 µg kg-1 or below with the exception of sulfachloropyridazine, tylosin, and tylvalosin. This demonstrates the effectiveness of feather analysis to detect antibiotic use to allow effective enforcement of antibiotic use and prevent the illegal, off-label, and nonregistered use of antibiotics.

Development and validation of a multiclass method for the determination of antibiotic residues in honey using liquid chromatography-tandem mass spectrometry.

A new, simple and fast method was developed for the determination of multi-class antibiotic residues in honey (sulfonamides, tetracyclines, macrolides, lincosamides and aminoglycosides). Separation and determination were carried out by LC-MS/MS. During sample preparation, various parameters affecting extraction efficiency were examined, including the type of solvent, pH, efficiencies of cleavage of N-glycosidic linkages by hydrochloric acid, ultrasonic extraction and its duration compared with shaking, along with dispersive SPE clean-up. Experiments with fortified samples demonstrated that 10 min of ultrasonic treatment with acidified methanol (HCl 2 M) followed by dispersive SPE clean-up with 50 mg PSA gave an effective sample preparation method for several classes of antibiotics in honey. Anhydroerythromycin A, erythromycin A enol ether and desmycosin were used as markers for the presence of erythromycin A and tylosin A in honey samples. The method was validated according to European Commission Decision (EC) No. 2002/657. The recoveries of analytes ranged from 85% to 111%. Repeatability and intra-laboratory reproducibility were < 20.6% and 26.8%, respectively. Decision limit (CCα) and detection capability (CCß) ranged from 6 to 9 µg kg-1 and from 7 to 13 µg kg-1, respectively, except for streptomycin and neomycin, which showed slightly higher CCα at 25 µg kg-1 and CCß at 34 µg kg-1. Finally, the method was applied to the honey test material 02270 through a FAPAS proficiency test (PT) for the determination of tetracyclines. PT results were obtained within a z-score range of ±2, proving that the validated method is suitable for routine analysis to ensure the quality of honey.

A Liquid Chromatography-Tandem Mass Spectrometry Method for the Detection of Antimicrobial Agents from Seven Classes in Calf Milk Replacers: Validation and Application.

Calf milk replacers are low-cost feeds that contain available, digestible protein. During their reconstitution, however, the addition of drugs, such as antibiotics, could make them a very simple route for illicit treatment for therapeutic, preventive, or growth-promoting purposes. We developed an HPLC-MS/MS method, preceded by a unique extraction step, able to identify 17 antibiotics from seven classes (penicillins, tetracyclines, fluoroquinolones, sulfonamides, cephalosporins, amphenicols, and lincosamides) in this matrix. Prior to solid phase extraction (SPE), the sample underwent deproteinization and defatting. The method was fully validated according to Commission Decision 2002/657/EC. Decision limits (CCα) and detection capability (CCß) were in the ranges of 0.13-1.26 and 0.15-1.47 ng/mL, respectively. Thirty-eight samples were finally analyzed, showing the occasional presence of marbofloxacin (six samples) and amoxicillin (one sample).

Performance characterization of a quantitative liquid chromatography-tandem mass spectrometric method for 12 macrolide and lincosamide antibiotics in salmon, shrimp and tilapia.

This paper describes an extension and performance characterization of a quantitative confirmatory multi-residue liquid chromatography-tandem mass spectrometric method for residues of macrolide and lincosamide antibiotics, originally validated for application to bovine kidney tissues, to tissues of salmon, shrimp and tilapia. The 12 analytes include clindamycin, erythromycin A, gamithromycin, josamycin, lincomycin, neospiramycin 1, oleandomycin, pirlimycin, spiramycin 1, tildipirosin, tilmicosin and tylosin A. The limit of detection was 0.5 µg/kg. Within-laboratory precision evaluated over the analytical range of 5.0-50.0 µg/kg ranged from 4 to 17%. The accuracy of the method ranged from 80 to 112%. Recoveries ranged from 47 to 99% with all but one recovery above 60%. This is the first report of a quantitative confirmatory method for gamithromycin, pirlimycin and tildipirosin in fish and shrimp.

Multiresidue analysis of antibiotics in food of animal origin using liquid chromatography-mass spectrometry.

Antibiotics are the most important drugs administered in veterinary medicine. Their use in food-producing animals may result in antibiotic residues in edible tissues, which are monitored to protect human and animal health, support the enforcement of regulations, provide toxicological assessment data, and resolve international trade issues. This chapter provides basic characterization of the most important classes of antibiotics used in food-producing animals (aminoglycosides, amphenicols, ß-lactams, macrolides and lincosamides, nitrofurans, quinolones, sulfonamides, and tetracyclines), along with examples of practical liquid chromatographic-(tandem) mass spectrometric methods for analysis of their residues in food matrices of animal origin. The focus is on multiresidue methods that are favored by regulatory and other food testing laboratories for their ability to analyze residues of multiple compounds in a time- and cost-effective way.

Heat treatment effects on the antimicrobial activity of macrolide and lincosamide antibiotics in milk.

Antibiotic residues in milk can cause serious problems for consumers and the dairy industry. Heat treatment of milk may diminish the antimicrobial activity of these antibiotic residues. This study analyzed the effect of milk processing (60 °C for 30 min, 120 °C for 20 min, and 140 °C for 10 s) on the antimicrobial activity of milk samples fortified with three concentrations of three macrolides (erythromycin: 20, 40 and 80 µg/liter; spiramycin: 100, 200, and 400 µg/liter; and tylosin: 500, 1,000, and 2,000 µg/liter) and one lincosamide (lincomycin: 1,000, 2,000, and 4,000 µg/liter). To measure the loss of antimicrobial activity, a bioassay based on the growth inhibition of Micrococcus luteus was done. The data were analyzed using a multiple linear regression model. The results indicate that treatment at 120 °C for 20 min produces inactivation percentages of 93% (erythromycin), 64% (spiramycin), 51% (tylosin), and 5% (lincomycin), while treatment at 140 °C for 10 s results in generally lower percentages (30% erythromycin, 35% spiramycin, 12% tylosin, and 5% lincomycin). The lowest loss or lowest reduction of antimicrobial activity (21% erythromycin and 13% spiramycin) was obtained by treatment at 60 °C for 30 min.

Simultaneous determination of polyether ionophores, macrolides and lincosamides in hen eggs by liquid chromatography-electrospray ionization tandem mass spectrometry using a simple solvent extraction.

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method was developed and validated for the determination of residues of 6 polyether ionophores (lasalocid, maduramicin, monensin, narasin, salinomycin, semduramicin), 3 macrolides (erythromycin, tylosin, clarithromycin) and 1 lincosamide (lincomycin) in eggs. Nigericin was used as qualitative internal standard. Samples were deproteinizated/extracted with acetonitrile without pH adjustments. Aliquots of the extracts were evaporated and reconstituted for injection in the instrument operated in positive multiple reaction monitoring (MRM) mode. The stability of the antibiotics and the intensity of the formed ions were considered in order to select a suitable solvent for the reconstitution of the obtained dry extracts. No clean-up steps were required and matrix effects were controlled by sample dilution, selection of appropriate chromatographic conditions and reduced injection volume. Good within-laboratory reproducibility was obtained, with relative standard deviations (RSD(R)) from 4.0 (semduramicin at 5 µgkg(-1)) to 18.6 (erythromycin at 25 µgkg(-1)) for the ionophores and macrolides. Lincomycin showed the least precise results, with a maximum RSD(R) of 20.2% at 75 µgkg(-1)). Satisfactory decision limits (CCα) and detection capabilities (CCß) were also attained. Method limits of detection (LODs) from 0.04 (salinomycin) to 1.6 µgkg(-1) (lincomycin) were achieved. Method limits of quantification (LOQs) were from 0.14 to 5.3 µgkg(-1) for the same drugs, respectively. All the LOQs, except that obtained for maduramicin were remarkably below the lowest validation level. The proposed method is suitable for routine application in commercial egg samples.

Effects of Swine manure on macrolide, lincosamide, and streptogramin B antimicrobial resistance in soils.

Current agricultural practices involve inclusion of antimicrobials in animal feed and result in manure containing antimicrobials and antimicrobial-resistant microorganisms. This work evaluated the effects of land application of swine manure on the levels of tetracycline, macrolide, and lincosamide antimicrobials and on macrolide, lincosamide, and streptogramin B (MLS(B)) resistance in field soil samples and laboratory soil batch tests. MLS(B) and tetracycline antimicrobials were quantified after solid-phase extraction using liquid chromatography-tandem mass spectrometry. The prevalence of the ribosomal modification responsible for MLS(B) resistance in the same samples was quantified using fluorescence in situ hybridization. Macrolide antimicrobials were not detected in soil samples, while tetracyclines were detected, suggesting that the latter compounds persist in soil. No significant differences in ribosomal methylation or presumed MLS(B) resistance were observed when amended and unamended field soils were compared, although a transient (<20-day) increase was observed in most batch tests. Clostridium cluster XIVa accounted for the largest fraction of resistant bacteria identified in amended soils. Overall, this study did not detect a persistent increase in the prevalence of MLS(B) resistance due to land application of treated swine manure.

Pilot survey of hen eggs consumed in the metropolitan area of Rio de Janeiro, Brazil, for polyether ionophores, macrolides and lincosamides residues.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which has recently been developed and validated, was used for the identification and quantification of polyether ionophore, macrolide and lincosamide residues in commercial eggs sold in the metropolitan area of Rio de Janeiro, Brazil. The method was applied to 100 samples and the results showed a high incidence of polyether ionophore residues (25%). Salinomycin was detected in 21% of samples, but only two non-compliant results (5.3 and 53 µg kg(-1)) were found if maximum limits (tolerances) established by European Union were adopted in Brazil and if a method decision limit (CCα) of 3.4 µg kg(-1) was considered. In 8% of analyzed samples, more than one studied coccidiostat was found. The lincosamide, lincomycin, and the macrolide, tylosin, were detected at trace levels in 4 and 1% of the samples, respectively. Lasalocid, clarithromycin and erythromycin were not found.

[Simultaneous determination of macrolide and lincosamide antibiotics in animal feeds by ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry].

A method for the simultaneous determination of six macrolide antibiotics (oleandomycin, erythromycin, kitasamycin, josamycin, roxithromycin and tylosin) and two lincosamide antibiotics (lincomycin and clindamycin) in animal feeds by ultra-performance liquid chromatography-electrospary ionization tandem mass spectrometry (UPLC-ESI-MS/MS) was developed. The macrolide and lincosamide antibiotics were extracted from the feeds with methanol followed by enrichment and clean-up with an Oasis HLB cartridge. The UPLC separation was performed on a Waters Acquity UPLC BEH C18 column by a gradient elution using 0.1% formic acid and acetonitrile as the mobile phase at a flow rate of 0.3 mL/min. The identification of eight drugs was carried out by positive electrospray ionization in multiple reaction monitoring (MRM) mode, and the quantification analysis was performed by external standard method. The calibration curves showed good linearity in the range of 1-100 microg/L. The average recoveries of the eight drugs from the feeds spiked at 1, 10 and 100 microg/kg levels were between 68.6% and 95.2%, and the relative standard deviations (RSD) were between 4.9% and 11.8%. The limits of quantification (LOQ) of the drugs in the feeds were 1 microg/kg. The method is simple, rapid, sensitive and suitable for the simultaneous determination of macrolide and lincosamide antibiotics in animal feeds.

[Determination of lincosamide residues in honey using high performance liquid chromatography-electrospray tandem mass spectrometry].

An analytical method for the determination of lincosamide antibiotic residues in honey was developed. In the procedure a solid-phase extraction for the isolation of lincomycin and clindamycin from diluted honey samples was employed. The residues of lincomycin and clindamycin were subsequently analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) coupled with electrospray tandem mass spectrometry. Mass spectral acquisition was applied with selective reaction monitoring of two diagnostic transition reactions. The qualitative analysis was based on the retention time, the precursor ion and two product ions, and the quantitation was carried out with intension of the characteristic ion m/z 126. The linear ranges were from 1.0 microg/L to 200 microg/L for lincomycin and clindamycin and the correlation coefficients (r2) were all greater than 0.996. The average recoveries for lincomycin and clindamycin ranged from 80% to 110% in replicate sets of honey samples spiked with the drug concentrations of 1.0, 5.0 and 20.0 microg/kg, and the relative standard deviations (RSDs) were less than 8% for intra-day and 15% for inter-day determinations. The method detection limit and quantitation limit were 0.1 micro/kg and 0.5 microg/kg, respectively. The method is simple and suitable for routine analysis.