Evaluation of the order of hierarchical structures for the calculation of method uncertainty using nested and other designs.

Several methods have been used to calculate method uncertainties using data from a previous publication. The major effort was to study the effect of changing the primary factor of the nested (hierarchical) design from concentration to matrix and then analyst. In the measurement of fluoroquinolones in honey, it was observed that when matrix was the primary factor the uncertainty values were generally slightly larger than when concentration or analyst were the primary factors. However, the differences are small, indicating the robustness of the nested design calculations. Two previously published methods of correcting for uncertainty due to intermediate precision were also examined and showed very little difference, confirming the robustness of the correction. A comparison was also made with previous non-nested designs, which tended to give lower calculated error values.

A new approach to the analysis of nicarbazin and ionophores in eggs by HPLC/MS/MS.

An HPLC/MS/MS method has been developed and validated for the quantification and confirmation of nicarbazin and ionophores (lasalocid, monensin, salinomycin, and narasin) in eggs. Nicarbazin is determined in the negative electrospray mode with a basic mobile phase that supports creation of negative ions. Consequently, our ability to maintain instrument sensitivity over time has significantly improved. The analysis of the ionophores is done in the positive electrospray mode using ammonium buffer for HPLC separation. Monitoring ammonium adduct parent ions resulted in enhanced sensitivity and better reproducibility of the ionophore analysis. The validation of this improved HPLC/MS/MS method for the detection of nicarbazin and the ionophores demonstrated excellent precision of below 10% RSD and lower LOD values (microg/kg) for nicarbazin (0.018), lasalocid (0.015), monensin (0.015), salinomycin (0.033), and narasin (0.039).

Quantitation of fluoroquinolones in honey using tandem mass spectrometry (LC-MS/MS): nested validation with two mass spectrometers.

A number of drugs in the quinolone and fluoroquinolone families, approved for veterinary treatment of food animals by various countries, may be used to treat bee diseases and thereby contaminate honey. An LC-MS/MS method has been developed for the quantification of the quinolones: flumequine, nalidixic acid, oxolinic acid, and pipemidic acid; and the fluoroquinolones ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, norfloxacin, ofloxacin, orbifloxacin, marbofloxacin, sarafloxacin, and sparfloxacin. A method-matched calibration curve is used with several internal standards, i.e., ciprofloxacin-d8, Iomefloxacin, and cinoxacin, to correct for the various types of honey matrices: white, light, medium, and dark colors. Enoxacin is added as an external recovery standard. The LOD values range from 0.05 microg/kg (ofloxacin) to 0.4 microg/kg (flumequine). The compounds are verified by LC-MS/MS retention times and ion ratios. Method uncertainty was determined using two separate analytical systems. The method has successfully measured the presence of norfloxacin in several samples of honey imported into Canada.

Quantitation and validation of macrolide endectocides in raw milk by negative ion electrospray MS/MS.

Macrolide endectocides (abamectin, doramectin, emamectin, eprinomectin, ivermectin, and moxidectin) are used to treat animals against a variety of parasites. They are approved for use with food animals, but require long withdrawal times. Two compounds, eprinomectin and moxidectin, are approved for use with lactating cattle and have established maximum residue limit values of 20 and 40 microg/kg, respectively. The remaining compounds may appear in milk if there is off-label or accidental use. This method is capable of quantitating and confirming the presence of six of the macrolide endectocides over the concentration range 1-60 microg/kg (parts per billion). Selamectin is used as the internal standard. The compounds are extracted with C18 solid-phase extraction under basic conditions and quantitated by negative ion LC-MS/MS using one selected reaction monitoring transition, with a second transition used for verification. The limits of detection were determined from between-day experiments as 0.14, 0.14, 0.18, 0.24, 0.25, and 0.25 microg/kg for abamectin, doramectin, emamectin, eprinomectin, ivermectin, and moxidectin, respectively. The detection capability CCbeta for eprinomectin was measured as 28.5 microg/kg, and 50.0 microg/kg for moxidectin using five milk matrices.

Ivermectin use and resulting milk residues on 4 Canadian dairy herds.

The Canadian gFARAD was contacted for milk withdrawal recommendations after multiple cases of topical ivermectin use in lactating dairy cows. The following 4 cases included pertinent milk residue information and illustrate the challenges faced by producers, veterinarians, and regulatory authorities when ivermectin use occurs in dairy cows.

Quantitation and validation of fluoroquinolones in eggs using liquid chromatography/tandem mass spectrometry.

Fluoroquinolone antibiotics are labeled for very limited veterinary use for treatment of some animals and pets, but they are not authorized for food animals in Canada, including egg-producing birds. Because they are approved for other animals, however, fluoroquinolones may appear in eggs through off-labeling or accidental use. This method is capable of quantitating and confirming the presence of 4 fluoroquinolones, ciprofloxacin, danofloxacin, enrofloxacin, and sarafloxacin, over the concentration range 1 to 60 microg/kg (ppb) using 2 internal standards, norfloxacin or lomefloxacin. The compounds are extracted with acidic acetonitrile, isolated using Oasis HLB solid-phase extraction, and quantitated by liquid chromatography/tandem mass spectrometry at 2 transitions. The limits of detection (microg/kg) were evaluated from between-day experiments as: ciprofloxacin, 0.22; danofloxacin (m/z 314), 0.32; enrofloxacin, 0.22; and sarafloxacin, 0.31. The values for the decision limit, CCalpha were 0.33, 0.36, 0.30, and 0.45 microg/kg, respectively.

Positive and negative electrospray LC-MS-MS methods for quantitation of the antiparasitic endectocide drugs, abamectin, doramectin, emamectin, eprinomectin, ivermectin, moxidectin and selamectin in milk.

Avermectin endectocides are used for the treatment of cattle against a variety of nematode and arthropod parasites, and consequently may appear in milk after normal or off-label use. The compounds abamectin, doramectin, and ivermectin, contain only C, H and O and may be expected to be detected by LC-MS in negative ion mode. The others contain nitrogen in addition and would be expected to be preferentially ionized in positive mode. The use of positive ion and negative ion methods with electrospray LC-MS-MS were compared. Using negative ion the compounds abamectin, doramectin, ivermectin, emamectin, eprinomectin, and moxidectin gave a curvilinear response and were quantified in raw milk by LC-MS-MS with a triethylamine-acetonitrile buffer over the concentration range 1-60 ppb (microg/kg) using selamectin as the internal standard. The limits of detection (LOD) were between 0.19 ppb (doramectin) and 0.38 ppb (emamectin). The compounds gave maximum sensitivity with positive ionisation from a formic acid-ammonium formate-acetonitrile buffer and were detected in milk (LC-MS-MS) also with a curvilinear response over the range 0.5-60 ppb. Although the positive ion signals were larger, with somewhat lower limits of detection (LOD between 0.06 ppb (doramectin) and 0.32 ppb (moxidectin) the negative ion procedure gave a more linear response and more consistent results. Comparison of spiked samples in the range 2-50 ppb showed a high degree of correlation between the two methods.