Marcos Fernández, Pablo López

Validation and quality control assignment

General information:
• Which is the Analyte considered?
The analytes to determine are β-lactams (ampicillin, amoxicillin…), cephalosporines and sulfonamides in
animal feed (object).

• Which is the Matrix considered?

The matrix that has been considered are deuterated compounds (amoxicillin-d4, sulfathiazole-d4, penicillin G-
d7 and penicillin V-d5), Oasis HLB (6 cc, 200 mg) cartridges, used for solid phase extraction, HPLC grade water,
methanol (MeOH), acetonitrile (ACN) and formic acid (98–100%) (FA) and hydromatrix.

• What is the aim of the study?

The main aims of this study are:
-To develop a multiresidue method based on PLE and SPE followed by LC–QLIT-MS/MS for the detection and
quantification of 18 antibacterials, including 7 penicillins, 4 cephalosporins and 7 sulfonamides in animal feed
even at small quantities.
-To validate this new analytical method taking into account the EU requirements.
-To check the applicability of the developed method by analysing real feed samples.

Techniques:
• Which are the Sampling technique and instrumentation used?
A representative sample obatained from bovine feed purchased from the Laboratori Agroalimentari of the
Generalitat de Cataluna (Cabrils, Spain) and ˜ stored in glass pots at room temperature until use feed samples
were grounded in a Multiquick/Minipimer professional MR 5000M mixer (Braun GmbH).

• Which is the Sample preparation technique selected?

The sample preparation techniques selected are pressurized liquid extraction (PLE) and solid phase extraction
(SPE).

• Which sample preparation parameters have been studied?

Marcos Fernández, Pablo López

Preservation (stored in glass pots at room temperature until use), concentration (different concentration
levels), extraction (with previous mentioned techniques) and clean-up ( In order to check whether the clean-up
step was really required or whether the desired results could be obtained without further sample manipulation, the PLE
extracts were filtered through 0.2 m or 0.45 m filters in order to remove any suspended material from the otherwise
fairly clean extracts.)

• Which is the Determination technique used?

Liquid chromatography-quadrupole-linear ion-trap mass spectrometry (LC-QLIT-MS/MS)

Validation of the analytical procedure:

• Which parameters have been evaluated?
Linearity, robustness (repeatability), sensitivity, precision, selectivity, LOD, LOQ and accuracy of the method.

• How have they been evaluated? Which samples? Number of replicates? Conditions?...
In order to evaluate the linearity in the tested matrix, nine levels of increasing concentrations of antibacterials from 25 to
300 ng/g were prepared in three different types of feed, errors were compensated using internal standards and mean
values.

For the robustness and the precision. The overall method repeatability was calculated as intra-day and inter-day
precision. Intra-day precision, is expressed as the average of the relative standard deviation (RSD %) of the areas
obtained for each analyte after the replicate (n = 6) analysis, during the same day, of three feed samples fortified at the
lowest available level (50 ng/g) with all 18 analytes and expressed as the RSD % between these six replicates. Inter-day
precision, was calculated as the percent relative standard deviation (RSD %) of the six replicate samples analysed during
three consecutive days.

The sensitivity is stimated by calculation of LOD range.

LODs and LOQs for the 18 antibacterials were calculated as three and ten times signal to noise ratio, respectively. The
calculated mean values are obtained from the analysis of fortified feed at levels of 50, 150 and 300 ng/g of all
substances. In order to compensate for the variations between different nature feed samples, the results reported
represent the average values obtained from the six replicate analysis (3 g feed) of three feed samples.

Selectivity was assured by utilizing a QTrap system in the MRM mode and obtaining single chromatographic peaks for
each of the SRM transitions. Identification and quantification using four identification points for each analyte, as required
by the EC, was achieved by monitoring two transition products for every precursor ion corresponding to each target
analyte in the MRM mode.

For the accuracy fortified samples were prepared by adding exact amounts of the appropriate working mixtures to 3 g of
three different feed samples. After fortification, the samples were allowed to equilibrate for about 20 h (overnight) at −2
◦C to −4 ◦C before extraction. Analyte recoveries were calculated from the peak areas obtained for each analyte (average
Marcos Fernández, Pablo López

of six replicates for each feed sample) as percentages of the peak areas obtained from the replicate (n = 3) analysis of
equivalent HPLC water spikes.

• What results have been achieved?

The validation showed high recoveries for all analytes (>71%), robustness (repeatability <8.5%) and high
sensitivity (LOD range: 0.12–4.99 ng/g). Application of the developed method to the analysis of 14 real feed
samples, revealed levels for penicillins ranging from 0.004 to 4.201 mg/kg and sulfonamides ranging between
0.018 and 1.683 mg/kg.
We think that there is a mistake in the results and therefore the LOD range is from 0.11-4.99 ng/g in regards of
table number 2. Value of LOD of Sulfadiazine.

Quality control:
• Which are the assays performed for quality control?
Instrumental blanks, extraction blanks, and full procedural blanks were analysed in the beginning and in
between fortified extracts. Instrument blanks were composed of MeOH and were analysed at the beginning of
the run. Extraction blanks consisted of HPLC water undergone SPE and analysed along with the samples.
Finally, full procedural blanks were prepared by three different types of feed sample extracted and analysed
after instrumental blanks.

Conclusions:
• What is the conclusion of this study?
All samples analysed contained at least one of the compounds under analysis.
This method is capable of detecting the low concentrations that could result from failure to comply to the
regulations or on-site contamination.