{"id":17747,"date":"2017-12-21T10:22:21","date_gmt":"2017-12-21T10:22:21","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=17747"},"modified":"2018-08-24T06:47:56","modified_gmt":"2018-08-24T06:47:56","slug":"development-and-validation-of-an-uplc-esi-msms-analytical-method-for-the-determination-of-streptomycin-and-dihydrostreptomycin-residues-in-honey","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol10no4\/development-and-validation-of-an-uplc-esi-msms-analytical-method-for-the-determination-of-streptomycin-and-dihydrostreptomycin-residues-in-honey\/","title":{"rendered":"Development and Validation of an UPLC-ESI-MS\/MS Analytical Method for the Determination of Streptomycin and Dihydrostreptomycin Residues in Honey"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Without any hesitation honey is the most recognized and well-known natural food produced by bees (Apis\u00a0<\/em>mellifera<\/em>) from nectar and honeydew (Cuili\u00a0et al<\/em>. 2016). It has at least 181 substances mainly fructose and glucose, and has minor amounts of proteins, enzymes, amino acids, minerals, trace elements, vitamins, aroma compounds and\u00a0polyphenol\u00a0(Alvarez-Suarez\u00a0et al.<\/em>\u00a02010;\u00a0De-Melo\u00a0et al.<\/em>\u00a02017). Its historic, cultural and economic significance make it the important beekeeping product. Bee keeping has become billion dollar industries in modern days, but due microbiological infestation, environmental, botanical and bad\u00a0apicultural\u00a0practices, honey bees and their combs gets effected. This leads to\u00a0contamination of honey and so inferior quality. Major contaminations of honey in\u00a0apicultural\u00a0produces are antibiotic and pesticide residue (Zhou\u00a0et al.<\/em>\u00a02014;\u00a0Zai\u00a0et al.<\/em>\u00a02013). However, presence of antibiotic residue are major concerns as antibiotic residue originating from agricultural usage or\u00a0 apicultural\u00a0practices can adversely influence public health due to\u00a0allergenic\u00a0and carcinogenic factors, and may give to bacterial resistance (Wassenaa\u00a0TM 2005).<\/p>\n

The occurrence of antibiotic residues in human foods, arising from its veterinary use is a cause of concern to consumers worldwide (Taokaenchan\u00a0and\u00a0Sangsrichan\u00a02010). Streptomycin (STR) is an\u00a0aminoglycoside\u00a0produced by\u00a0Streptomyces\u00a0<\/em>griseus<\/em>\u00a0<\/em>strains and\u00a0dihydrostreptomycin\u00a0(Di-STR) is the product of\u00a0<\/em>its catalytic hydrogenation.\u00a0 STR\u00a0is protein synthesis inhibitors and in spite of their toxicity, it is widely used in veterinary medicine for treatment of aerobic gram-negative bacteria (Oliveira\u00a0et al.<\/em>\u00a02009;\u00a0Horie\u00a0et al.<\/em>\u00a02004).\u00a0STR\u00a0is commonly used in apiculture for the prophylactic treatment or control of bacterial brood diseases such as European foul brood and American foul brood disease (Victoria\u00a0et al.<\/em>\u00a02007; Pena\u00a0et al.<\/em>\u00a02009). Due to these instances, contamination of antibiotics in honey is unavoidable. In the German market, it was found that 21% of 183 honey sample contained streptomycin residues. 1.7% samples found to be positive for streptomycin, sulfonamides, tetracycline, chloramphenicol, nitrofurans, tylosin and quinolones out of 3855 samples, consequently samples was a noncompliant as per \u00a0European Standards (Al-Waili et al.<\/em> 2012). During 2000-2001, streptomycin\u00a0detected\u00a0in 4 samples out of 248 honey sample. Honey samples collected during the peak flowering season in southern part of Tamil\u00a0Nadu, India\u00a0reported\u00a0to have streptomycin residue in the range of 4\u201317 ng\/kg (Solomon\u00a0et al.<\/em>\u00a02006).<\/p>\n

Centre for Science and Environments (CSE\u2019s) Pollution Monitoring Lab, or PML found that out of 12 branded honey samples, 11 samples was contaminated with six antibiotics and the tested samples were brought randomly from various markets of Delhi in July 2009. 10 samples were Indian brands and 2 samples were imported brands (CSE, Delhi, India). Presence of such antibiotic residue in honeys is unfavorable for human consumption. High concentrations of streptomycin may produce ototoxicity and nephrotoxic effects. However, regular consumption of STR at low concentrations in foods may also cause allergies, destroy intestinal flora and cause resistance to certain microorganisms (Cara et al. <\/em>2013; Gacia et al.<\/em> 2015). High levels of antibiotic residue in honey exported from India to EU and US have been reported by Agricultural Processed Food Product Export Development Agency (APEDA) from 2005 onwards (Al-Waili et al.<\/em> 2012).<\/p>\n

So far, there are no\u00a0maximum\u00a0residue limits (MRLs) for antibiotic residues in honey. Therefore,\u00a0the presence of\u00a0veterinary drugs in honey is not authorized. The European Union Reference Laboratories (EU-RLs) provide recommended concentrations (RCs) for the control of non-authorized substances in honey as presented in Table 1\u00a0<\/strong>in order to improve and harmonize\u00a0the performance of\u00a0the monitoring analytical methods (Hawari\u00a0et al.<\/em>\u00a02017;\u00a0Mahmoudi\u00a0et al.<\/em>\u00a02014).<\/p>\n

Table 1: Tolerance levels (\u03bcg kg-1<\/sup>) for veterinary drugs in honey in several countries<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\u00a0<\/strong><\/td>\nEU-RLs<\/strong><\/td>\nIndia<\/strong><\/td>\nCanada<\/strong><\/td>\nAustralia<\/strong><\/td>\nSwitzerland<\/strong><\/td>\n<\/tr>\n
Classes<\/strong><\/td>\nRCs<\/strong><\/td>\nMRLs<\/strong><\/td>\nMRLs<\/strong><\/td>\nMRLs<\/strong><\/td>\nMRLs<\/strong><\/td>\n<\/tr>\n
Streptomycin<\/td>\n40<\/td>\n10<\/td>\n125<\/td>\n–<\/td>\n10<\/td>\n<\/tr>\n
Tetracyclines<\/td>\n20<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
Tetracycline<\/td>\n–<\/td>\n10<\/td>\n250<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
Chlortetracycline<\/td>\n–<\/td>\n10<\/td>\n100<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
Oxytetracycline<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n300<\/td>\n–<\/td>\n<\/tr>\n
Sulphonamide<\/td>\n50<\/td>\n10<\/td>\n100<\/td>\n–<\/td>\n50<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Various methods have been\u00a0developed and validated for the analysis of antibiotic residues in human food. The selection of method is dependent on the\u00a0objective of the analysis, availability and the required sensitivity for that particular analysis.\u00a0Chromatographic\u00a0techniques coupled with mass spectrometry have turn out to be very popular in recent years, most notably ULTRA performance liquid chromatography\u2013mass spectrometry (UPLC-MS) (Kivrak\u00a0et al.<\/em>\u00a02016;\u00a0Tamosiunas\u00a0and\u00a0Padarauskas\u00a02008). Method development for\u00a0STR\u00a0and\u00a0Di-STR\u00a0becomes challenging because, these antibiotics are highly polar organic basic compounds. Moreover, it shows practically no retention in reversed phase liquid chromatography, except an ion-pairing reagent added to the mobile phase, also considering the suitable concentration to\u00a0 reduce ionization suppression (Kujawski\u00a0and\u00a0Namiesnik\u00a02008).<\/p>\n

The aim of the present study was to develop a simple, precise and economical analytical method for determination of\u00a0STR\u00a0in honey, applying solid phase extraction (SPE) and\u00a0UPLC-ESI-MS\/MS determination. To enhance the accuracy and precision of the analytical method,\u00a0validation\u00a0was done\u00a0in compliant of\u00a0European Commission (EC) Decision 2002\/657\/EC. Finally, the method applied for the quantification of\u00a0STR\u00a0and\u00a0Di-STR\u00a0residue in different honey samples.<\/p>\n

Material and Methods<\/strong><\/p>\n

Chemical and Reagents<\/strong><\/p>\n

STR\u00a0and\u00a0Di-STR\u00a0purchased\u00a0from Sigma Aldrich (St. Louis, MO, USA), SPE Cartridge (Waters\u00a0Sep-pak\u00a0Vac\u00a06cc\u00a0Accell\u00a0plus CM), Acetic acid, Milli-Q water, Acetonitrile,\u00a0 Aquity\u00a0UPLC\u00a0BEH\u00a0HILIC\u00a01.7 \u03bcm, 2.1×100 mm column from waters (Waters Co., Milford, MA, USA),\u00a0Ammonium\u00a0formate,\u00a0 Formic acid. LC\u2013MS grade Acetonitrile\u00a0 purchased from Merck\u00a0Millipore.\u00a010mg\u00a0standards weighed with 0.1mg sensitivity into 10 ml volumetric flask and dissolved into water:\u00a0acetonitrile\u00a0(80:20). \u00a0Concentration of stock standards\u00a0calculated\u00a0considering their purity percentages.<\/p>\n

\u00a0Sample Collection<\/strong><\/p>\n

21 honey samples\u00a0purchased\u00a0from the local markets of New Delhi and\u00a0Gurgaon, India during the month of July – October in the year 2016. Samples\u00a0stored\u00a0at room temperature in the dark until analysis. Selected honey samples\u00a0checked\u00a0for targeted antibiotics using proposed method, samples found\u00a0to be\u00a0free from target antibiotics\u00a0 were considered\u00a0as blank.<\/p>\n

Sample Extraction<\/strong><\/p>\n

10g\u00a0honey sample\u00a0weighed\u00a0into 50 ml centrifuge tube and mixed with\u00a025 ml Milli-Q water to dissolve honey. Furthermore 25 ml Milli-Q water added to make up solution up to 50 ml. After that, samples\u00a0filtered through\u00a0Whattman\u00a0No-1 filter paper and filtered\u00a0sample solution loaded into\u00a0SPE cartridge and passed at 2 drops per sec. 5 ml elution\u00a0solvent 2% acetic acid in water and\u00a0acetonitrile\u00a0(80:20) used for\u00a0elution\u00a0and collected into 5 ml volumetric flask. Required volume were adjusted with Milli-Q water and filtered into\u00a0UPLC\u00a0vial for analysis.<\/p>\n

Instrumentation<\/strong><\/p>\n

Chromatographic\u00a0analysis\u00a0\u00a0performed on a\u00a0UPLC-MS\/MS (Waters\u00a0Acquity\u00a0Ultra Performance LC-MS\/MS, Waters Co., and Milford, MA, USA) equipment consists of a Waters\u00a0acquity\u00a0ultra performance liquid chromatography with a Waters column manager, binary system manager, sample manager coupled to a Waters\u00a0XEVO\u00a0TQD\u00a0triple quadruple mass spectrometer equipped with\u00a0electrospray\u00a0ionization (ESI). An\u00a0acquity\u00a0UPLC\u00a0BEH\u00a0Hillic\u00a0column 1.7\u00a0\u03bcm X 2.1 mm X 100 mm used for\u00a0the determination of\u00a0STR\u00a0and\u00a0Di-STR. The mobile phase was (A) Ammonium\u00a0formate\u00a0in water having \u00a0pH 2.5 adjusted with formic acid\u00a0 and\u00a0 (B) Formic acid\u00a0Acetonitrile\u00a0in the ratio (90:10), Injection flow\u00a05\u03bcL, flow rate 400\u00a0\u03bcL\/min, Column oven temp 40\u00b0C. <\/em>The gradient program was: 0 – 0.1 (A) 10% and (B) 90 %, 0.1 – 2.5 minute (A) 90% and (B) 10%, 2.5 – 6.0 (A) 10% and (B) 90 %.\u00a0The analysis of samples\u00a0carried in the positive\u00a0ESI-MS-MS\u00a0ion mode.<\/p>\n

Mass Spectrometry<\/strong><\/p>\n

MS\/MS\u00a0parameters\u00a0and precursor-product ions of\u00a0STR\u00a0and\u00a0Di-STR\u00a0tuned by direct infusion in the\u00a0SRM\u00a0mode and 0.40 ml\/min flow rate of the mobile phase (A) and (B).\u00a0MS\/MS detector parameters\u00a0presented in Table 2.<\/p>\n

Table 2: MS\/MS detector parameters<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
Ionization mode<\/em><\/strong><\/td>\nPositive<\/em><\/strong><\/td>\n<\/tr>\n
MS Aquire time<\/em><\/td>\n6.0 minute<\/em><\/td>\n<\/tr>\n
Capillary voltage<\/em><\/td>\n3.8kV<\/em><\/td>\n<\/tr>\n
Source temperature<\/em><\/td>\n150<\/em>\u00b0C<\/em><\/td>\n<\/tr>\n
Disolvation temperature<\/em><\/td>\n550<\/em>\u00b0C<\/em><\/td>\n<\/tr>\n
Ion energy<\/em><\/td>\n0.50<\/em><\/td>\n<\/tr>\n
Cone gas flow<\/em><\/td>\n25 L\/Hr<\/em><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Method Validation<\/strong><\/p>\n

The UPLC\u2013MS\/MS method\u00a0validated\u00a0according to 2002\/657\/EC guidelines. UPLC\u2013MS\/MS identification of antibiotic residues\u00a0verified\u00a0during the course of validation study by monitoring relative retention times, ion recognition (signal-to-noise ratio) and relative ion intensities.\u00a0UPLC-MS\/MS identification criteria were set out in the legislation and verified throughout\u00a0validation of the\u00a0method. In this study, various validation parameters such as selectivity, linearity, recovery (accuracy), repeatability (precision), limit of quantification (LoQ), decision limit (CC\u03b1), detection capacity (CC\u03b2), ruggedness\u00a0validated\u00a0to\u00a0evaluate\u00a0performance of\u00a0the developed method.<\/p>\n

Results and Discussion<\/strong><\/p>\n

An\u00a0accurate, simple, fast and\u00a0cost effective\u00a0UPLC\u2013ESI- MS\/MS method\u00a0developed\u00a0and validated as per European\u00a0Commission Decision 2002\/657\/EC\u00a0for\u00a0determination of\u00a0STR\u00a0and\u00a0Di-STR\u00a0residue in honey samples. Subsequently,\u00a0quantification of\u00a0STR\u00a0and\u00a0Di-STR\u00a0achieved\u00a0by means of validated method in honey samples collected from the local markets of New Delhi &\u00a0Gurgaon, India.<\/p>\n

UPLC-ESI-MS\/MS Method Development<\/strong><\/p>\n

To carry out this experiment\u00a0Aquity\u00a0UPLC\u00a0BEH\u00a0Hilic\u00a0column\u00a0used\u00a0with\u00a020mM\u00a0ammonium\u00a0formate\u00a0in Milli-Q water having pH 2.5 adjusted with formic acid and\u00a0acetonitrile\u00a0as mobile phase. The gradient program in the analysis under the conditions described in the\u00a0methodology\u00a0section allows\u00a0separation of\u00a0STR\u00a0and\u00a0Di-STR\u00a0with good resolution. The mean retention time for\u00a0STR\u00a0and\u00a0Di-STR\u00a0was obtained 1.70 minutes as presented in Fig. 1. However, Area for the blank sample spiked with 10.0\u00a0\u03bcg\u00a0kg-1<\/sup>\u00a0was illustrated\u00a0in Table 3. \u00a0Previous studies reported that,\u00a0analysis of\u00a0STR\u00a0and\u00a0Di-STR\u00a0by means of LC-MS\/MS using mobile phase A as (water with 0.05% formic acid) and B (Acetonitrile\u00a0with 0.05% formic acid) and the\u00a0coloum\u00a0Hilic\u00a0Atlantis (150 x 2.1 mm, 3\u00a0\u00b5m\u00a0particle size) and the\u00a0Hilic\u00a0MonoChrom\u00a05 MS from Varian (150 x 2 mm), each with adequate guard\u00a0were used\u00a0(Bohm\u00a0et al.\u00a0<\/em>2012). Retention time obtained in this study was at 8.0 minutes, which was higher than our retention time 1.70 minutes.\u00a0Granja\u00a0et al.<\/em>\u00a02009 reported determination of streptomycin using column Gemini 5\u00a0\u00b5m\u00a0C18<\/sub>\u00a0(50mm\u00d72mm) and\u00a05mM\u00a0\u00a0heptafluorobutiric\u00a0acid\/acetonitrile\u00a0(85:15) as the mobile phase at a flow rate of 200\u00a0\u00b5L\u00a0min\u22121. Retention time obtained in this study\u00a0was at 5.40 minutes. This\u00a0was also higher than our retention times. From the retention point of view the present\u00a0study have\u00a0advantage over former one.<\/p>\n

MS\/MS technique allows for mass analysis to take place in a sequential\u00a0manner\u00a0in different regions of the instrument. Triple quadruple system follows the tandem in space arrangement due to ionization, primary mass\u00a0selection, collision induced dissociation, and mass analysis of fragments produced during collision induced dissociation and detection occurring in separate segments of the instrument. Optimized Multiple Reaction Monitoring (MRM) transition of the validated method\u00a0has been presented\u00a0in Table 4.<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 1: Chromatogram showing multiple reactions monitoring for streptomycin and Dihydrostreptomycin<\/strong><\/p>\n

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Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Table 3: Selectivity for validated method of streptomycin and dihydrostreptomycin<\/strong><\/p>\n\n\n\n\n\n\n\n
Compound\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 1\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 2\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 3\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 4\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 6\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Mean\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 SD\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 % RSD<\/strong><\/td>\n<\/tr>\n
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STR<\/strong><\/td>\n

RT<\/td>\n1.70<\/td>\n1.70<\/td>\n1.70<\/td>\n1.71<\/td>\n1.70<\/td>\n1.71<\/td>\n1.70<\/td>\n0.004<\/td>\n0.217<\/td>\n<\/tr>\n
Area<\/td>\n128<\/td>\n137<\/td>\n139<\/td>\n144<\/td>\n141<\/td>\n137<\/td>\n138<\/td>\n5.4<\/td>\n3.9<\/td>\n<\/tr>\n
 <\/p>\n

Di-STR<\/strong><\/td>\n

RT<\/td>\n1.70<\/td>\n1.70<\/td>\n1.70<\/td>\n1.70<\/td>\n1.70<\/td>\n1.70<\/td>\n1.70<\/td>\n0.000<\/td>\n0.000<\/td>\n<\/tr>\n
Area<\/td>\n201<\/td>\n231<\/td>\n217<\/td>\n220<\/td>\n224<\/td>\n208<\/td>\n217<\/td>\n10.9<\/td>\n5.0<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Table 4: Summary of multiple reactions monitoring for analytes detected in positive ionization mode<\/strong><\/p>\n\n\n\n\n\n\n\n
Compound<\/strong><\/td>\nParent ion<\/strong><\/td>\nDaughter ion<\/strong><\/td>\nCone Voltage<\/strong><\/td>\nRT<\/strong><\/td>\nDwell Time (Sec)<\/strong><\/td>\nCollision<\/strong><\/p>\n

Energy<\/strong><\/td>\n<\/tr>\n

STR<\/td>\n582.4<\/td>\n246.09<\/td>\n80<\/td>\n1.70<\/td>\n0.1<\/td>\n35<\/td>\n<\/tr>\n
263.13<\/td>\n80<\/td>\n1.70<\/td>\n0.1<\/td>\n30<\/td>\n<\/tr>\n
Di-STR<\/td>\n584.5<\/td>\n246.10<\/td>\n80<\/td>\n1.70<\/td>\n0.1<\/td>\n35<\/td>\n<\/tr>\n
263.14<\/td>\n80<\/td>\n1.70<\/td>\n0.1<\/td>\n30<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

RT- Retention time<\/p>\n

Method Validation<\/strong><\/p>\n

Validation was carried out in accordance with the procedures outlined in Europe Commission Decision 2002\/657\/EC covering specificity, linearity, accuracy, precision, Limit of Quantification (LoQ), Decision limits (CC\u03b1) and Decision capability (CC\u03b2). The ruggedness of the method was demonstrated as an ongoing basis through the use of it, to analyze various honey sample collected from local markets of New Delhi & Gurgaon, India during 2016. The values identified for these parameters were all within Europe Commission Decision 2002\/657\/EC.<\/p>\n

Specificity\/ Selectivity<\/strong><\/p>\n

The developed method was checked for specifity by preparing 7 different honey matrix blank samples and injected into the UPLC-MS\/MS system. The analysis was performed and from the chromatogram & mass spectra. We found that, no significant peaks with S\/N (signal to noise) ratios and no interferences at the retention time of our interest of analytes as presented in Fig. 2 and the method was specific.<\/p>\n\n\n\n
\u00a0\"Vol10No4_Dev_Sye_fig2\"<\/td>\nFigure 2: Specificity chromatogram of blank honey (A) sample and spiked sample (B)\u00a0<\/strong>Linearity<\/strong><\/p>\n

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Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

A matrix calibration curves were constructed for analyte in the sample. A sufficient number of standards were used to adequately define the relationship between concentration and response. We constructed a matrix spiked calibration curve with the 6 different concentration ranges from 2.0 \u03bcg kg-1<\/sup>, 5.0 \u03bcg kg-1<\/sup>, 10.0 \u03bcg kg-1<\/sup>, 15.0 \u03bcg kg-1<\/sup>, 20.0 \u03bcg kg-1<\/sup> and 50.0 \u03bcg kg-1<\/sup> of the anlayte. A calibration curves were evaluated between the Area vs<\/em> Concentrations of the analyte and the r2<\/sup> was calculated 0.994. This was acceptable as per Commission Decision 2002\/657\/EC.<\/p>\n

Accuracy\/Recovery<\/strong><\/p>\n

Recovery of an anlayte was obtained from the known concentration added to the sample matrix and recovered. To evaluate the recovery 7 blank honey samples were taken each spiked with STR and Di-STR at 2.0 \u00b5g kg-1<\/sup>, 5.0 \u00b5g kg-1<\/sup>, 10.0 \u00b5g kg-1<\/sup>, 20.0 \u00b5g kg-1<\/sup> and 50.0 \u00b5g kg-1<\/sup>. Recoveries of seven spiked samples were presented in Table 5 and Table 6. The recovery results were observed in acceptable range of 80-110 %.<\/p>\n

Table 5: Showing spiked concentration versus estimated concentration<\/strong><\/p>\n\n\n\n\n\n\n\n\n
\u00a0<\/strong><\/p>\n

Spike Concentration<\/strong><\/td>\n

Streptomycin\u00a0 (\u00b5g kg-1<\/sup>)<\/strong><\/td>\n<\/tr>\n
2.0<\/strong><\/td>\n5.0<\/strong><\/td>\n10.0<\/strong><\/td>\n20<\/strong><\/td>\n50.0<\/strong><\/td>\n<\/tr>\n
Mean (7 sample)<\/td>\n2.07<\/td>\n4.64<\/td>\n9.32<\/td>\n19.82<\/td>\n50.16<\/td>\n<\/tr>\n
SD<\/td>\n0.25<\/td>\n0.49<\/td>\n1.08<\/td>\n0.33<\/td>\n0.76<\/td>\n<\/tr>\n
RSD %<\/td>\n5.7<\/td>\n11.73<\/td>\n11.58<\/td>\n6.71<\/td>\n14.92<\/td>\n<\/tr>\n
Recovery %<\/td>\n85.9<\/td>\n82.7<\/td>\n93.2<\/td>\n96.8<\/td>\n99.5<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

SD- Standard deviation, RSD- Relative standard deviation<\/p>\n

Table 6: Showing spiked concentration versus estimated concentration<\/strong><\/p>\n\n\n\n\n\n\n\n\n
\u00a0<\/strong><\/p>\n

Spike Concentration<\/strong><\/td>\n

Dihydrostreptomycin\u00a0 (\u00b5g kg-1<\/sup>)<\/strong><\/td>\n<\/td>\n<\/tr>\n
2.0<\/strong><\/td>\n5.0<\/strong><\/td>\n10.0<\/strong><\/td>\n20<\/strong><\/td>\n50.0<\/strong><\/td>\n<\/tr>\n
Mean (7 sample)<\/td>\n1.96<\/td>\n4.51<\/td>\n10.08<\/td>\n19.48<\/td>\n51.11<\/td>\n<\/tr>\n
SD<\/td>\n0.45<\/td>\n0.23<\/td>\n0.39<\/td>\n0.50<\/td>\n1.16<\/td>\n<\/tr>\n
RSD %<\/td>\n10.09<\/td>\n5.27<\/td>\n3.92<\/td>\n10.05<\/td>\n8.02<\/td>\n<\/tr>\n
Recovery %<\/td>\n91.7<\/td>\n88.2<\/td>\n99.9<\/td>\n91.6<\/td>\n93.2<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

SD- Standard deviation, RSD- Relative standard deviation<\/p>\n

Repeatability (Precision)<\/strong><\/p>\n

Repeatability of STR and Di-STR in honey was performed by taking blank honey samples fortified with STR and Di-STR at 2.0 \u00b5g kg-1<\/sup>, 5.0 \u00b5g kg-1<\/sup>, 10.0 \u00b5g kg-1<\/sup>, 20.0 \u00b5g kg-1<\/sup> and 50.0 \u00b5g kg-1<\/sup>. For each level, analysis was performed with 7 replicates as per the EC guidelines. Same method was repeated in two other different days and Coefficient of variation (CV) % and Mean recovery (MR) % were calculated as shown in Table 7.<\/p>\n

Table 7: showing spiked concentration at different levels with % mean recovery and % coefficient of variation<\/strong><\/p>\n\n\n\n\n\n\n
\u00a0<\/strong><\/p>\n

Analyte<\/strong><\/td>\n

2<\/strong> \u00b5g kg-1<\/sup><\/strong><\/td>\n5 \u00b5g kg-1<\/sup><\/strong>\u00a0<\/strong><\/td>\n10 \u00b5g kg-1<\/sup><\/strong><\/td>\n20 \u00b5g kg-1<\/sup><\/strong><\/td>\n50<\/strong> \u00b5g kg-1<\/sup><\/strong><\/td>\n<\/tr>\n
MR%<\/strong><\/td>\nCV%<\/strong><\/td>\nMR%<\/strong><\/td>\nCV%<\/strong><\/td>\nMR%<\/strong><\/td>\nCV%<\/strong><\/td>\nMR%<\/strong><\/td>\nCV%<\/strong><\/td>\nMR%<\/strong><\/td>\nCV%<\/strong><\/td>\n<\/tr>\n
STR<\/td>\n93.8<\/td>\n4.1<\/td>\n93.30<\/td>\n13.0<\/td>\n94.40<\/td>\n9.9<\/td>\n101.5<\/td>\n3.9<\/td>\n96.0<\/td>\n10.4<\/td>\n<\/tr>\n
Di-STR<\/td>\n98.7<\/td>\n3.0<\/td>\n92.40<\/td>\n7.0<\/td>\n97.60<\/td>\n4.8<\/td>\n94.9<\/td>\n2.2<\/td>\n94.60<\/td>\n10.5<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

MR- Mean Recovery\u00a0\u00a0\u00a0 CV- Coefficient of variation<\/p>\n

Limit of Quantification (LoQ)<\/strong><\/p>\n

The minimum concentration of the anlayte that can be quantified with acceptable accuracy and precision.\u00a0 LoQ was evaluated, 7 Honey samples were spiked with STR and Di-STR at concentration of 2.0 \u03bcg kg-1<\/sup> and determination was performed 3 times, Recovery percentage and Relative standard deviation were calculated as presented in Table 8. The recovery obtained was in the range of 92- 104%, however RSD was below 11%. So, we conclude our LoQ as 0.15 \u03bcg kg-1<\/sup>. Values calculated for LoQ was within the EC regulation.<\/p>\n

Table 8: showing Limit of quantification of streptomycin and dihydrostreptomycin with spiked concentration<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
Conc. Spiked\u00a0\u00a0\u00a0\u00a0\u00a0 (\u03bcg kg-1<\/sup>)<\/strong><\/td>\nConc. Recovered (\u03bcg kg-1<\/sup>) STR<\/strong><\/td>\nMean<\/strong><\/td>\n%RSD<\/strong><\/td>\nRecover%<\/strong><\/td>\n<\/tr>\n
1<\/strong><\/td>\n2<\/strong><\/td>\n3<\/strong><\/td>\n4<\/strong><\/td>\n5<\/strong><\/td>\n6<\/strong><\/td>\n7<\/strong><\/td>\n<\/tr>\n
2.0<\/td>\n1.99<\/td>\n1.82<\/td>\n1.90<\/td>\n1.92<\/td>\n2.03<\/td>\n2.04<\/td>\n1.84<\/td>\n1.95<\/td>\n4.3<\/td>\n97.5<\/td>\n<\/tr>\n
2.0<\/td>\n1.82<\/td>\n1.67<\/td>\n1.81<\/td>\n1.72<\/td>\n1.83<\/td>\n1.64<\/td>\n1.74<\/td>\n1.75<\/td>\n4.9<\/td>\n87.4<\/td>\n<\/tr>\n
2.0<\/td>\n2.06<\/td>\n2.02<\/td>\n2.19<\/td>\n2.04<\/td>\n2.07<\/td>\n2.00<\/td>\n2.12<\/td>\n2.08<\/td>\n2.9<\/td>\n104.2<\/td>\n<\/tr>\n
Conc. Spiked\u00a0\u00a0\u00a0\u00a0\u00a0 (\u03bcg kg-1<\/sup>)<\/strong><\/td>\nConc. Recovered (\u03bcg kg-1<\/sup>) Di- STR<\/strong><\/td>\nMean<\/strong><\/td>\n%RSD<\/strong><\/td>\nRecover%<\/strong><\/td>\n<\/tr>\n
1<\/strong><\/td>\n2<\/strong><\/td>\n3<\/strong><\/td>\n4<\/strong><\/td>\n5<\/strong><\/td>\n6<\/strong><\/td>\n7<\/strong><\/td>\n<\/tr>\n
2.0<\/td>\n1.98<\/td>\n2.06<\/td>\n2.14<\/td>\n1.96<\/td>\n1.97<\/td>\n2.19<\/td>\n1.82<\/td>\n2.05<\/td>\n4.9<\/td>\n102.5<\/td>\n<\/tr>\n
2.0<\/td>\n1.92<\/td>\n1.89<\/td>\n1.93<\/td>\n1.93<\/td>\n1.83<\/td>\n1.87<\/td>\n1.82<\/td>\n1.90<\/td>\n2.2<\/td>\n94.8<\/td>\n<\/tr>\n
2.0<\/td>\n1.94<\/td>\n1.91<\/td>\n1.97<\/td>\n1.90<\/td>\n1.91<\/td>\n2.03<\/td>\n1.95<\/td>\n1.94<\/td>\n2.6<\/td>\n97.2<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Decision limits (CC\u03b1), Decision Capability (CC\u03b2)<\/strong><\/p>\n

The CC\u03b1 is the lowest concentration level of the analyte that can be detected in a sample with a chance of 1% of a false positive decision whereas CC\u03b2 is the smallest content of the analyte, which can be detected in a sample with a chance of 5% of false negative decision. In compliance with the decision 2002\/657\/EC concerning the performance of the methods, CC\u03b1 and CC\u03b2 were elaborated using blank honey samples fortified with (2.0, 5.0, 10.0, 20.0 and 50.0 \u03bcg kg-1<\/sup>). CC\u03b1 and CC\u03b2 values obtained for STR and Di-STR was 11.68 \u03bcg kg-1<\/sup>,11.27 \u03bcg kg-1<\/sup> and 13.36 \u03bcg kg-1<\/sup>, 13.36 \u03bcg kg-1<\/sup>respectively. Both CC\u03b1 and CC\u03b2 were presented in Table 9. This was found to be in compliance with EC Decision.<\/p>\n

Table 9: showing validation parameters for CAP<\/strong><\/p>\n\n\n\n\n\n
Compounds<\/strong><\/td>\nLoQ (\u03bcg kg-1<\/sup>)<\/strong><\/td>\nDecision Limit (CC\u03b1) (\u03bcg kg-1<\/sup>)<\/strong><\/td>\nDetection Capability (CC\u03b2) (\u03bcg kg-1<\/sup>)<\/strong><\/td>\n<\/tr>\n
STR<\/td>\n2.0<\/td>\n11.68<\/td>\n13.36<\/td>\n<\/tr>\n
Di-STR<\/td>\n2.0<\/td>\n11.27<\/td>\n12.55<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Application of the Developed Method to Market Samples<\/strong><\/p>\n

After validation of the analytical methodology, the applicability of the method was evaluated by analyzing 21 honey samples collected from local market of New Delhi and Gurgaon, India. These samples were processed as described in material & method section. In 21\u00a0honey samples, none of them were detected for STR and Di-STR as presents in Fig. 3. The identification criteria were based on the presence of chromatographic peaks for both transitions (qualifier and quantifier) at the same retention time. Quantification was carried out using calibration standards that were prepared by spiking blank samples before extraction with the analytes at different specific concentration levels used for validation.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 3: Total ion chromatogram of streptomycin and dihydrostreptomycin in honey sample<\/strong><\/p>\n

 <\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Conclusion<\/strong><\/p>\n

The proposed method\u00a0developed\u00a0and validated for\u00a0determination of\u00a0STR\u00a0and its derivatives\u00a0Di-STR\u00a0in honey as per Commission Decision 2002\/657\/EC. The\u00a0objective\u00a0of this study was to develop a\u00a0time saving,\u00a0cost effective, precise and sensitive method for determination of\u00a0STR\u00a0and\u00a0Di-STR.\u00a0\u00a0The sample preparation procedure was simple, which is critical for a rapid analysis that makes procedure less susceptible to\u00a0analyte\u00a0losses and as a result an adequate extraction with recovery (82\u2013102% for\u00a0STR, 88\u2013102% for\u00a0Di-STR). The method linearity was acceptable with correlation coefficients above 0.994 for both the\u00a0analytes\u00a0in the linear range from 2\u201350\u00a0\u03bcg kg-1.\u00a0\u00a0All the obtained data fulfills the requirements laid down by European Commission Decision 2002\/657\/EC.\u00a0\u00a0The method allowed for the monitoring of parent ion and two fragment ions for each\u00a0analyte, which attains the purpose of confirming\u00a0identity of\u00a0the\u00a0analytes. The validation parameters showed that the method developed was adequate for the quantification and confirmation of\u00a0STR\u00a0and\u00a0Di-STR\u00a0residue in honey at concentration lower than the recognized RC 40\u00a0\u03bcg kg-1 led by European Commission Decision 2002\/657\/EC.<\/p>\n

Acknowledgements<\/strong><\/p>\n

We are thankful to the Department of Food technology, F.E.I.S., Hamdard University, New Delhi and Department of Food technology, JJT University, Rajasthan for providing facilities to carrying out the present study<\/p>\n

Conflict of Interest<\/strong><\/p>\n

There is no conflict of interest.<\/p>\n

Funding Sources<\/strong><\/p>\n

There was no funding source to carry out this study.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Cuili M, Spano N, Pilo M.I, Sanna G. Recent advances in the analysis of phenolic compounds in unifloral honeys. Molecules.<\/em>\u00a0\u00a02016;21:451.
    \n    CrossRef<\/a><\/li>\n
  2. Alvarez-Suarez J.M, Tulipani S,\u00a0 Romandini S, Bertoli E,\u00a0 Battino M.\u00a0 Contribution of honey in nutrition and human health: a review. Mediterranean Journal of Nutrition and \u00a0Metabolism.\u00a0<\/em>2010;3:15\u201323.
    \n    CrossRef<\/a><\/li>\n
  3. De-Melo A.A.M,\u00a0 Almeida-Muradian D.L.B, Sancho M.T, Pascual-Mate A. Composition and properties of Apis mellifera<\/em> honey: A review. Journal of Apicultural Research.<\/em>\u00a02017.<\/li>\n
  4. Zhou J, Yao L, Li Y, Chen L, Wu L, Zhao J. Floral classification of honey using liquid chromatography\u2013diode array detection\u2013tandem mass spectrometry and chemometric analysis. Food Chemistry.\u00a0<\/em>2014;<\/em>145: 941\u2013949.
    \n    CrossRef<\/a><\/li>\n
  5. Zai I.U.M, Rehman K, Hussain A,\u00a0 Detection and quantification of antibiotics residues in honey samples by chromatographic techniques. Middle-East Journal\u00a0 Scientific Research.\u00a0<\/em>\u00a02013;14(5):683-687.<\/li>\n
  6. Wassenaa T.M. The use of antimicrobial agents in veterinary medicine and implications for human health. Critical Reviews Microbiology.<\/em>\u00a02005;31:155-169.
    \n    CrossRef<\/a><\/li>\n
  7. Taokaenchan N, Sangsrichan S. HPLC-Fluorescence detection method for quantitative determination of tetracycline antibiotic residues in honey. NU Science Journal.<\/em>\u00a02010;6(2):147\u2013155.<\/li>\n
  8. Oliveira R.C.D, Paschoal J.A.R, Sismotto M, Airoldi F.P.D.S, Reyes F.G.R. Development and validation of an LC\u2013APCI-MS\u2013MS analytical method for the determination of streptomycin and dihydrostreptomycin residues in milk. Journal Chromatographic Science.\u00a0<\/em>2009;47:757-761.
    \n    CrossRef<\/a><\/li>\n
  9. Horie M, Saito H, Natori T, Nagata J, Nakazawa H. Determination of streptomycin and dihydrostreptomycin in honey by liquid chromatography\u2013electrospray mass spectrometry. Journal of Liquid Chromatography & Related Technologies. <\/em>2004;<\/em>27(5):863\u2013874.
    \n    CrossRef<\/a><\/li>\n
  10. Victoria B, Marghitas L.A, Dezmirean D, Moise A, Bobis O, Maghear O. Optimization of HPLC method for quantifying tetracycline residue in honey. Bulletin USAMV-CN.<\/em>\u00a0 2007;63:186-190.<\/li>\n
  11. Pena A,\u00a0 Pelantova\u00a0 N,\u00a0 Lino C.M,\u00a0 Silveira M.I.N, Solich P. Validation of an analytical methodology for determination of oxytetracycline and tetracycline residues in honey by HPLC with fluorescence detection. \u00a0Journal Agricultural and Food Chemistry.\u00a0\u00a0<\/em>2005;<\/em>53:3784-3788.
    \n    CrossRef<\/a><\/li>\n
  12. Al-Waili N, Salom K, Al-Ghamdi A, Ansari M.J. Antibiotic, pesticide, and microbial contaminants of honey: human health hazards. The Science World Journal.<\/em>\u00a02012;1-9.
    \n    CrossRef<\/a><\/li>\n
  13. Solomon R, Satheeja S, Vimalan J. Prevalence of antibiotics in nectar and honey in South Tamil Nadu, India. Integra Biosciences.\u00a0<\/em>2006;<\/em>10: 163-167.<\/li>\n
  14. http:\/\/www.cseindia.org\/content\/cse-lab-study-busting-myth-about-%E2%80%98pure-and-natural%E2%80%99-honey<\/li>\n
  15. Cara M.C, Dumitrel G, Glevitzky M,\u00a0 Mischie C, Silaghi-Perju D.\u00a0 \u00a0Thermal degradation of streptomycin residues in honey during storage. Food Technology and Biotechnology.\u00a0<\/em>2013;<\/em>51(3):429\u2013433.<\/li>\n
  16. Gacic M, Bilandzic N, Sipusic D.I, Petrovic M, Kos B, Vahcic N, Suskovic J. Degradation of oxytetracycline, streptomycin, sulphathiazole and chloramphenicol residues in different types of honey. Food Technology and Biotechnology. <\/em>2015;<\/em>53(2):154\u2013162.<\/li>\n
  17. Hawari K.E, Mokh S, Doumyati S, Iskandarani M.A, Verdon E. Development and validation of a multiclass method for the determination of antibiotic residues in honey using liquid chromatography tandem mass spectrometry.\u00a0Food Additives & Contaminants: Part. A.\u00a0<\/em>2016;34(4).<\/li>\n
  18. Mahmoudi R, Moosavy M, Norian R, Kazemi S, Nadari M.R.A, Mardani K.\u00a0 Detection of oxytetracycline residues in honey samples using ELISA and HPLC methods. Pharmaceutical sciences.\u00a0\u00a0<\/em>2014;<\/em>19(4):145-150.<\/li>\n
  19. Kivrak I, Kivrak S, Harmandar M. Development of a rapid method for the determination of antibiotic residues in honey using UPLC-ESI-MS\/MS. Food Science and Technology<\/em> (Campinas).<\/em>\u00a02016;36(1):90-96.
    \n    CrossRef<\/a><\/li>\n
  20. Tamosiunas V, Padarauskas A. Comparison of LC and UPLC coupled to MS\u2013MS for the determination of sulfonamides in egg and honey. Chromatographia.\u00a0\u00a0<\/em>2008;<\/em>67:783\u2013788.
    \n    CrossRef<\/a><\/li>\n
  21. Kujawski M.W, Namiesnik J. Challenges in preparing honey samples for chromatographic determination of contaminants and trace residues. Trends in\u00a0 Analytical Chemistry. <\/em>2008;<\/em>27(9):785-793.
    \n    CrossRef<\/a><\/li>\n
  22. Bohm D.A, Stachel C.S, Gowik P. Confirmatory method for the determination of streptomycin and dihydrostreptomycin in honey by LC\u2013MS\/MS. Food Additives & Contaminants: Part A.<\/em>\u00a0 2012;29(2): 189\u2013196.
    \n    CrossRef<\/a><\/li>\n
  23. Granjaa \u00a0R.H.M.M, Nino A.M.M, \u00a0Zucchetti R.A.M, Nino R.E.M, Patel R, Salerno A.G. \u00a0Determination of streptomycin residues in honey by liquid chromatography\u2013tandem mass spectrometry. Analytica Chimica Acta.\u00a0<\/em>\u00a02009;637:64\u201367.
    \n    CrossRef<\/a><\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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