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CFSAN/Office of Plant and Dairy Foods
September 29, 2006
Method Developed by
Florida Department of Agriculture and Consumer Services
Preparation and LC/MS/MS Analysis of Honey for Fluoroquinolone Residues
Honey is dissolved in water, and then any fluoroquinolone residues are extracted into acetonitrile after acidification. Water is removed from the acetonitrile by passing the extract through a sodium sulfate column. The extract is concentrated, then assayed using LC-MS/MS.
This method will allow the determination and confirmation of enrofloxacin and ciprofloxacin in honey at concentrations above 2.5 ppb.
All work with organic solvents must be done in a chemical fume hood. Personal protective gear is required, including safety glasses, solvent resistant gloves and laboratory coat.
Disposal glassware is used wherever possible to eliminate the possibility of cross contamination. Great care must be taken to clean non disposable glassware in the procedure.
Note: Other quantities of these reagents may be prepared by scaling the proportions appropriately.
Time 0 min: | 95% A, 5% B | flow = 200 µL/min |
Time 15 min: | 5% A, 95% B | flow = 200 µL/min |
Time 25 min: | 5% A, 95% B | flow = 200 µL/min |
Time 25.1 min: | 95% A, 5% B | flow = 500 µL/min |
Time 30.0 min: | 95% A, 5% B | flow = 500 µL/min |
Time 30.1 min: | 95% A, 5% B | flow = 200 µL/min |
Time 32.1 min: | 95% A, 5% B | flow = 200 µL/min |
Ciprofloxacin:
332>288 | transition in MS/MS (used for quantification) |
332>288>268 | transition in MS^3 (major qualitative ion transition) |
332>288>245 | transition in MS^3 (major qualitative ion transition) |
Retention time (approximate): 10.5 minutes
Enrofloxacin:
360>316 | transition in MS/MS (used for quantification) |
360>316>245 | transition in MS^3 (major qualitative ion transition) |
360>316>288 | transition in MS^3 (minor qualitative ion transition) |
Retention time (approximate): 10.9 minutes
Note: MS/MS on the ion trap yields one primary fragment ion and some smaller less abundant peaks for both ciprofloxacin and enrofloxacin. An additional stage of MS analysis (MS^3) is performed on the primary fragment ion from the MS/MS experiment and yields sufficient points for confirmation.
Note: Other processing files may be needed based on the type of curve, number of points, or type of calibration performed.
Alternately, standard concentrations that represent the equivalent sample ng/g level of the target compound may be utilized in the data software with a dilution factor of 1.0. The curve is a linear fit, not forced through zero. If needed, a 1/x weighting may be used to more accurately quantitate low concentrations. Acceptable curves have correlation coefficients of 0.99 or greater. All quantitative sample responses must fall within the calibration curve.
Individual standards of enrofloxacin and ciprofloxacin are prepared in a solution of 16/84% acetonitrile/acetic acid (2%) by the standards section. These standards are each prepared at levels of 0.05, 0.1, 0.2, 0.5, and 1.0 ng/uL. These standards are stored in amber disposable vials in the freezer at -10°C. Standards should be prepared fresh for each batch of analyses.
Preparation of the standards for analysis of honey samples is as follows:
* Note - honey extract is a final extract (i.e. ready for instrumental analysis). It is generated by taking negative control honey and extracting it per this SOP. The reference matrix utilized for the method validation was tupelo honey.
1.25 ng/mL mixed working standard-in-matrix (2.5 ng/g sample equivalent)
2.5 ng/mL mixed working standard-in-matrix (5.0 ng/g sample equivalent)
5.0 ng/mL mixed working standard-in-matrix (10.0 ng/g sample equivalent)
12.5 ng/mL mixed working standard-in-matrix (25.0 ng/g sample equivalent)
25 ng/mL mixed working standard-in-matrix (50.0 ng/g sample equivalent).
Attachment A: Instrument Method
Attachment B: Example of a Tune File
Attachment C: Example of an Extracted Ion Chromatogram
Attachment D: Example of an Analytical Run Sequence
Attachment E: Quantification using Standard Additions
Attachment F: Typical Method
Recoveries versus External Standards in Tupelo Honey
REVISION HISTORY:
Rev | Date | Description | Author |
1 | 09-15-06 | FDACS method CR 405 Revision 4 dated 09/01/06 formatted for FDA use. | P. Kijak |
2 | 09-29-06 | Corrected limit in scope to 2.5 ppb, converted rpm to RCF | P. Kijak |
Instrument Method: FLUROQUINOLONE_CE_HIFLOW.meth
Agilent1100 AutoSampler
Drawing speed (µL/min): 200
Ejecting speed (µL/min) : 200
Needle draw position offset (mm) : 0.0
Injection volume (µL) : 10.0
Wash vial: 100
Wash Cycle: 1
Wash Stroke: 20.0
Analysis stop time (min): 32.10
Post run time (min) : 0.00
Timed events:
Time | Contact Number | Contact State |
---|---|---|
0.00(min) |
Agilent1100 Binary Pump
Solvent A: H2O/0.1% FORMIC
Solvent B: ACN/0.1% FORMIC
Minimum pressure limit (bar): 0.0
Maximum pressure limit (bar): 400.0
Post run time (min) : 0.00
Gradient program:
Time | Flow Rate | Composition |
---|---|---|
0.00(min) | 0.20(mL/min) | A=95.0% B=5.0% |
15.00(min) | 0.20(mL/min) | A=5.0% B=95.0% |
25.00(min) | 0.20(mL/min) | A=5.0% B=95.0% |
25.10 (min) | 0.50 (mL/min) | A=95.0% B=5.0% |
30.00(min) | 0.50(mL/min) | A=95.0% B=5.0% |
30.10(min) | 0.20 (mL/min) | A=95.0% B=5.0% |
32.10(min) | 0.20(mL/min) | A=95.0% B=5.0% |
Agilent1100 Heater
Oven: On
Separate Mode: On
Post run time (min): 0:00
Time | Left Temperature | Right Temperature | Valve Position |
---|---|---|---|
0.00(min) | 40.00(C) | 40.00(C) | 1 |
32.10(min) | 40.00(C) | 40.00(C) | 1 |
LTQ Instrument Method
Creator: LTQ
Last modified: 9/1/2006 by LTQ
MS Run Time (min) : 32.10
Sequence override of method parameters not enabled.
Divert Valve: in use during run
Divert Time (min) ============ |
Valve State ======== |
---|---|
0.00 | To Waste |
5.05 | To Source |
15.05 | To Waste |
Contact Closure: not used during run
Syringe Pump: not used during run
MS Detector Settings:
Additional Microscans:
MS2 0
MS3 0
MS4 0
MS5 0
MS6 0
MS7 0
MS8 0
MS9 0
MS10 0
Segment 1 Information
Duration (min) : 32.l0
Number of Scan Events: 2
Tune Method: STATESCREEN_122804
Scan Event Details:
1: ITMS + c norm !corona !pi · (332.0)->o(90.0-342.0)
MS/MS: CE 35.0% Q 0.250 Time 30.000 IsoW 3.0
2: ITMS + c norm !corona !pi · (360.0)->o(95.0-370.0)
MS/MS: CE 35.0% Q 0.250 Time 30.000 IsoW 3.0
Custom Data Dependent Settings:
Not enabled
510612E
STD HALFACTIONX_#1
Segment : 1
Capillary Temp (C) : | 300.00 |
APCI Vaporizer Temp (C) : | 0.00 |
Sheath Gas Flow () : | 50.00 |
Aux Gas Flow () : | 10.00 |
Sweep Gas Flow () : | 15.00 |
Source Type : | ESI |
Injection Waveforms : | Off |
Zoom AGC Target : | 1000.00 |
Full AGC Target : | 30000.00 |
SIM AGC Target : | 10000.00 |
MSn AGC Target : | 10000.00 |
Source Voltage (kV) : | 4.00 |
Source Current (µA) : | 100.00 |
Capillary Voltage (V) : | 9.00 |
Tube Lens (V) : | 100.00 |
Skimmer Off set (V) : | 0.00 |
Multipole RF Amplifier (Vp-p) : | 400.00 |
Multipole 00 Offset (V) : | -5.00 |
InterMultipole Lens 0 Voltage (V) : | -4.50 |
Multipole 0 Offset (V) : | -5.25 |
InterMultipole Lens 1 Voltage (V) : | -10.00 |
Gate Lens Offset (V) : | 0.00 |
Multipole 1 Offset (V) : | -6.50 |
Front Lens (V) : | -6.00 |
Zoom Micro Scans : | 1 |
Zoom Max Ion Time (ms) : | 50.00 |
Full Micro Scans : | 1 |
Full Max Ion Time (ms) : | 50.00 |
SIM Micro Scans: 1 | |
SIM Max Ion Time (ms) : | 50.00 |
MSn Micro Scans : | 1 |
MSn Max Ion Time (ms) : | 200.00 |
Data File Sample Name: STD_1XACTION_#2
Sample Type: Unknown
Acquistion Date: 08/15/06 22:48:35
Run Time (min): 32.10
Vial: 2
Injection Volume (µL): 10.00
Multiplier: 1.00
Comments: 510611E
Instrument Method: FLUROQUINOLONE_CE_HIFLOW.meth
Processing Method: None
Current Data Path: RUN#1_MSMS
Expected RT | RT | Name | Area | Height | Calculated Amount | Specified Amount | Units |
---|---|---|---|---|---|---|---|
9.68 | 9.68 | CIPROFLOXACIN | 11613 | 1762 | 5.212 | N/A | |
10.22 | 10.24 | ENROFLOXACIN | 24312 | 3991 | 4.727 | N/A |
Position | File Name | Inj Vol | Sample Type | Level | Dil Factor |
---|---|---|---|---|---|
1 | EQUIL01 | 10 | Unknown | 1 | |
1 | EQUIL02 | 10 | Unknown | 1 | |
1 | EQUIL03 | 10 | Unknown | 1 | |
1 | EQUIL04 | 10 | Unknown | 1 | |
1 | STD_2.5_#1 | 10 | Std Bracket | 2.5 PPB | 1 |
2 | STD_5_#1 | 10 | Std Bracket | 5 PPB | 1 |
3 | STD_10_#1 | 10 | Std Bracket | 10 PPB | 1 |
4 | STD_25_#1 | 10 | Std Bracket | 25 PPB | 1 |
5 | STD_50_#1 | 10 | Std Bracket | 50 PPB | 1 |
6 | Instrument Blank#1 | 10 | Unknown | 1 | |
7 | Control Honey Extract | 10 | Unknown | 1 | |
8 | Control Honey Extract | 10 | Unknown | 1 | |
9 | Fortified Honey Extract | 10 | Unknown | 1 | |
10 | Fortified Honey Extract | 10 | Unknown | 1 | |
11 | Sample | 10 | Unknown | 1 | |
12 | Sample | 10 | Unknown | 1 | |
1 | STD_2.5_#2 | 10 | Std Bracket | 2.5 PPB | 1 |
2 | STD_5_#2 | 10 | Std Bracket | 5 PPB | 1 |
3 | STD_10_#2 | 10 | Std Bracket | 10 PPB | 1 |
4 | STD_25_#2 | 10 | Std Bracket | 25 PPB | 1 |
5 | STD_50_#2 | 10 | Std Bracket | 50 PPB | 1 |
6 | Instrument Blank#2 | 10 | Unknown | 1 | |
13 | Sample | 10 | Unknown | 1 | |
14 | Sample | 10 | Unknown | 1 | |
15 | Sample | 10 | Unknown | 1 | |
16 | Sample | 10 | Unknown | 1 | |
17 | Sample | 10 | Unknown | 1 | |
18 | Sample | 10 | Unknown | 1 | |
19 | Sample | 10 | Unknown | 1 | |
20 | Sample | 10 | Unknown | 1 | |
1 | STD_2.5_#3 | 10 | Unknown | 1 | |
2 | STD_5_#3 | 10 | Unknown | 1 | |
3 | STD_10_#3 | 10 | Unknown | 1 | |
4 | STD_25_#3 | 10 | Unknown | 1 | |
5 | STD_50_#3 | 10 | Unknown | 1 |
Standard levels reflect concentration of target analyte in sample.
Action level = 5.0 ng/g
Quantification using standard additions may be necessary to improve accuracy in cases where the ion suppression or enhancement effects in a sample matrix are not adequately compensated for by the use of external standards prepared in the reference matrix. This technique utilizes known amounts of the target analyte added to the extracted sample so as to compensate for ion suppression or enhancement effects. Such effects have been observed when some other types of honey matrices were compared with the honey matrix used for the initial validation. Prior to standard additions, it will be necessary to make a rough estimate of the concentration of the target analyte in the sample using external standards as listed in sections 5.6.3. The procedure outlined in this attachment may then be utilized for the standard additions quantification. Variations of this procedure may also be necessary depending on the nature of the sample and the concentration of the target analyte. Adequate documentation of the standard additions experiment is vital.
One mL aliquots of sample extract are placed in each of three 2 mL size autosampler vials. These are labeled A, B, and C. Vials (B) and (C) are spiked with known amounts of the target analyte to produce signal counts roughly double (B vial), and triple (C vial) the counts present in the zero level sample (A vial). The volume of standard solution added to the (B) and (C) sample vials should be equal. Therefore, the use of standard solutions at the desired concentrations will be required. The same volume of blank solvent (solvent the spikes are prepared in) should be added to the (A) vial. Thus, A, B and C extracts are diluted to the same volume which simplifies the calculations necessary to obtain a final sample concentration. The target additions of standard to vials (B) and (C) are made based on estimates from the external standard quantification. These will only be estimates and may result in the signal counts in the (B) and (C) vials which do not provide reasonable detector response or regression and quantification. Additional estimates and repeated experiments may be necessary.
Once the sample extracts are spiked, each is analyzed and the area counts recorded for the target analyte in each of the A, B, and C vials. These values are then plotted vs the concentrations of the additions in each vial using a linear regression fit with the area counts plotted on the vertical axis (Y) and the concentrations of the additions plotted on the horizontal axis (X). Note: vial (A) has a concentration of (0) as no standard was added to this sample. The equation is then solved for (X) when (Y) equals zero. The absolute value of (X) is then corrected for the dilution resulting from the addition of the standard and solvent to vials A, B, and C. This corrected value is then multiplied by the concentration of sample by weight (grams solids analyzed) in the final extract (5.0 mL/2.5 gm) to obtain the final concentration of analyte in the sample. For example, if samples in vial A, B, and C are all diluted with 100 ul of standard or blank solvent then the correction value is (1.1/1.0) or 1.1 . This value would be multiplied by the value obtained from the curve and then by a factor of (5.0/2.5) or 2 to arrive at the final concentration of analyte in the sample in ng/g. If the extracts are diluted prior to the standard additions experiment this must also be factored into the final calculation.
Example of estimating the concentration of a target analyte in final extract from external standard quantification:
If a sample is calculated to contain a level of 5 ng/g of target analyte, then the concentration present in the final extract is as follows:
(5 ng/g) x (2.5 g) = 12.5 ng of target in final volume of 5 mL = 2.5 ng/mL in final extract.
To approximately double this concentration it is necessary to place 2.5 ng of target in the 1 mL aliquot of final extract used for the standard addition experiment. A standard with a concentration level of 0.05 ng/ul would require 50 ul of standard be pipeted into the 1 mL aliquot for an actual addition concentration of 2.5 ng/1.05 mL or 2.38 ng/mL. This would be sufficient to approximately double the concentration in the sample. The exact value of 2.38 would be plotted on the horizontal axis (X) vs the area counts obtained for this sample when measured by the analytical instrument.
Important points:
The fit of the linear regression curve should be greater than 0.99
All samples should be diluted to the same volume for simplification of the calculations.
Fortification Level (ppb) | Average Recovery (%) | Standard Deviation | CV (%) | Number of Samples |
---|---|---|---|---|
2.5 | 69 | 2 | 3 | 3 |
5.0 | 74 | 7 | 9 | 21 |
10 | 66 | 11 | 16 | 5 |
100 | 68 | 5 | 7 | 5 |
Fortification Level (ppb) | Average Recovery (%) | Standard Deviation | CV (%) | Number of Samples |
---|---|---|---|---|
2.5 | 95 | 3 | 3 | 3 |
5.0 | 90 | 7 | 8 | 21 |
10 | 85 | 16 | 19 | 5 |
100 | 90 | 3 | 3 | 5 |
Chemical Name: enrofloxacin
IUPAC International Chemical Identifier: