U. S. Food and Drug Administration
Laboratory Information Bulletin No. 4289
August 2002

The Laboratory Information Bulletin is a tool for the rapid dissemination of laboratory methods (or information) which appear to work. It may not report completed scientific work. The user must assure him/herself by appropriate validation procedures that LIB methods and techniques are reliable and accurate for his/her intended use. Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.

Scope

There are limited reports of the analysis of CAP and other phenicols in food from animal origin substances using electrospray LC/MS (3). Several others government (4,5) methods have also been reported, but are not published in the open literature. Our laboratory has been working with these compounds for many years. The traditional approach to the determination and confirmation of these compounds is isolation from tissue or fluids using liquid/liquid extraction, derivatization with silylating agents to form volatile derivatives, and analysis by GC/ECD and/or GC/MS with negative chemical ion detection (6-8).

Recently we have developed confirmatory methods for phenicols residues in shrimp and honey (9,10) using an ion trap LC/MS with an electrospray interface. This LIB describes the application of that method to the confirmation of chloramphenicol in crawfish tissue. Due to the fact that crawfish is a very dirty matrix some modification of the extraction procedure was required.

This data shows that this method is suitable for the confirmation of CAP in this matrix.

1. Roybal, J.E. "Chloramphenicol and Related Drugs" in Analytical Procedures for Drug Residues in Food of Animal Origin (1998) ed, S.B. Turnipseed and A.R. Long, Science Technology System, W. Sacramento, CA pp. 227-260.
2. http://www.fst.rdg.ac.uk/foodlaw/news/eu-02031.htm
3. Hormazabal, Y J. Liq Chromatogr. & Related Technique
4. Canadian Food Inspection Agency (CFIA), Dartmouth Laboratory Draft method: Analysis of Florfenicol, Florfenicol Amine, Thiamphenicol and Chloramphenicol in Fish, Shellfish and Crustaceans (2002)
5. Florida Chemical Residue Laboratories, Florida Department of Agriculture and Consumer Services, Preparation and Analysis of Chloramphenicol in Shrimp. (2002)
6. Pfenning, A.P., Roybal, J.E., Rupp, H.S., Turnipseed, S.B., Gonzales, S.A., Hurlbut, J.A. (2000) J.AOAC Int. 83, 26.
7. Pfenning, A.P., Madson, M.R., Roybal, J.E, Turnipseed, S.B., Gonzales, S.A., Hurlbut, J.A., Salmon, G.D (1998) J.AOAC Int. 81, 714.
8. Kijak, P.J (1994) J.AOAC. Int. 77, 34.
9. Turnipseed, S., Burns, C., Storey, J., Lee, R., Pfenning, A. (2002)FDA/ORA/DFS Laboratory Information Bulletin #4281
10. Pfenning, A., Turnipseed, S., Roybal, J., Burns, C., Storey, J., Madson, M., Lee, R., (2002)FDA/ORA/DFS Laboratory Information Bulletin #4284

Principles

1. Extraction
   
   Ten grams of crawfish composite is extracted with 20 mL ethyl acetate/ammonium hydroxide homogenized and centrifuged. The extraction steps are repeated and the ethyl acetate/ammonium hydroxide layers are evaporated to dryness. Thirty mL water is added to the flask, sonicated and followed by hexane defatting steps. The aqueous phase is passed through a series of SPE columns. The analyte is eluted off the final SPE with methanol. The methanol is evaporated to dryness. The extracts are reconstituted into a small volume of 0.1% formic acid and filtered into LC vials.
   
   
2. Mass Spectral Analysis
   
   A. Qualitative Confirmation
   
   The qualitative confirmation of phenicols in crawfish is based on unique mass spectral characteristics of these compounds as evaluated by established guidelines (11,12). One unique aspect of these compounds is the fact that they contain two chlorine atoms, thus giving rise to unique isotopic patterns. In order to take advantage of this fact, the MS² spectra is obtained not only from the parent ion ([M-H]-, but also from the corresponding M+2 (³⁵Cl³⁷Cl) isotope peak. For example, in the MS² spectra of CAP ([M-H]- pair m/z 321/323) the predominant ion is m/z 194 which corresponds to [M- H-(NH₂COCCl₂H)]^-. Also present in this spectra are the ions m/z 176 [m/z 194 - (H₂O)]^- (15%), 249 [M-H-(2HCl)]^- (30%), and 257 [M-H-(HCOCl)]^- (25%). These ions are also present in the MS² spectra of m/z 323, although the peak at 257 is split (into peaks of approximately equal abundance) between ions at m/z 257 and 259, indicating the loss of one chlorine atom (either ³⁵Cl or ³⁷Cl) from the ³⁵Cl³⁷Cl parent ion.

Reagents

Solvents: Distilled-in glass, pesticide-grade, hexane, ethyl acetate (EtOAC), acetonitrile (ACN) isopropanol (IPA), methanol (MeOH).
Formic acid used to prepare the mobile phase was purchased from Baker (88%).
Solid-phase extraction columns:

• C₁₈: Varian Bond Elut 6 cc/500 mg
• PRS: Varian Bond-Elut LRC-PRS 500mg.

Syringe filters:

• 4 mm syringe filter 0.45 µm, PFTE. Phenomenex P/N AFO-0422
• OR Gelman PVDF Acrodisc LC13 filter 0.45 µm.

Ammonium hydroxide (assay ca. 30% as NH₃).
Glacial acetic acid, LC grade.

Equipment

1. Ion Trap LC/MS: The instrument used was a Finnegan LCQ DECA Ion Trap Mass Spectrometer coupled to a modular Spectrasystem LC system. The components of the LC system include a SCM1000 degasser, P4000 LC pump, AS3000 autosampler, and a UV6000LP UV/VIS detector. The software used was Xcaliber Version 1.2.
2. LC Column: The LC Column was an Xterra phenyl (2.1 x 100 mm, 3.5 µ, Waters Corp. P/N 186001180). Other phenyl columns would also be acceptable. In this laboratory an Inertsil phenyl (2 x 150 mm, 5 µ, Phenomenex Corp. P/N 0301-150X020) was also tested during method development. If other columns are used, the acquisition program may need to be adjusted to account for shift in the retention time of CAP.
3. Other:
   Tissue disrupter --High speed shearing tool, i.e. tissuemizer, of a diameter < 20 mm.
   Rotoevaporator: with ice trap and water bath set at 50°C
   Nitrogen evaporator: 12-sample nitrogen evaporator, with 50°C water bath
   Plasticware: 50 mL and 15 mL disposable, conical polypropylene with screw cap
   Glassware: pear shape flask, Pasteur pipettes

Procedures

1. Standard Preparation
   
   The compounds were purchased or obtained from: Chloramphenicol (USP).

   Fortification Standards. For fortification of crawfish, individual stock solutions of drug at 1000 µg/mL (1000 ng/µL) were made up in acetonitrile. A combined intermediate standard solution (10 ng/µL) was made by pipetting 1 mL of each individual stock solution into 100 mL volumetric flask and diluting to volume with acetonitrile. Prepare fortification standards, as applicable: Pipet 0.5, 0.2, or 0.1 mL combined standard solution into 10 mL volumetric and dilute to volume with acetonitrile for 5, 2, and 1 ppb fortification standards, respectively.
   
   MS Standards For MS analysis, stock solutions of drug at 100 µg/mL (100 ng/µL) were made up in methanol. A mixed intermediate standard (1 ng/µL of each drug) was made by diluting 500 µL of each stock solution to 50 mL with 0.1% formic acid.
   
   Working LC/MS Standards. As applicable, LC/MS standards were made as follows:
   

   µL of intermediate standard	µL of 0.1% Formic Acid	[ng/µL]	equivalent in crawfish (ppb)*
   1000	4000	0.2	5
   400	4600	0.08	2
   200	4800	0.04	1
   100	4900	0.02	0.5

   * Assuming 10 g of crawfish is processed and final extract volume is 250 µL.

   
   
   Stability: Working LC/MS standard are stable for at least one week.

2. Sample Preparation

   Control Samples. At least one control (matrix blank) sample should be run with every set of samples.
   
   Fortified Samples. At least two fortified samples should be run with every set of incurred or unknown samples. The concentration of the fortified sample should be in the range of 1-5 ppb.
   
   Incurred Samples. Were not evaluated during method development.
   
   Sample Compositing. Crawfish are normally sold commercially precooked, peeled and frozen. For this reason, all analyses performed in this method were with precooked crawfish. Place crawfish meat in blender, and blend with dry ice with pulsed action until contents are uniform.

3. Sample Extraction
   
   Accurately weigh about 10.0 g of blended crawfish composite into a 50 mL P/P centrifuge tube. (If spiking control crawfish, add 100 µL of the desired concentration of Standard Solution to completely thawed 10 g blank composite and allow to sit at room temperature for at least 20 minutes before proceeding.) Add 20 mL of extraction solution (EtOAC:NH₄OH, 98:2) homogenize with tissue disrupter until the entire mass is broken up (about 30 sec). Centrifuge for 5 minutes at 4000 RPM at approximately 5°C. Pour into 100 mL P-S flask. Repeat extraction twice more, combinin g the extracts in the 100mL P-S flask. Add 10 mL IPA, to prevent bumping and roto-evaporate at 50-55°C to dryness. Ensure that there is no residual aroma of ethyl acetate or ammonium hydroxide. If necessary, add a few mLs of IPA, vortex and roto-evaporate to dryness. There will remain an oily residue. Add 30 mL H₂O, vortex, sonicate 4 min, adjust pH (<4.6) with approximately 0.4mL of 0.1% acetic acid. Filter aqueous through glass microfiber filter into a 50 mL P/P centrifuge tube. After the liquid has drained through the filter, rinse the filter with enough water to raise the level in the 50 mL P/P centrifuge tube to 30 mL. Discard the P-S flask. Do not rinse the flask with hexane. Add 10 mL of hexane to P/P centrifuge tube; shake vigorously for about 20 seconds. Centrifuge @ 4000 RPM at 5°C for 3 min, aspirate upper hexane layer and discard. Repeat hexane defatting steps one more time with an additiona l 10 mL portion of hexane each time and discard the hexane each time. Condition each PRS and C₁₈ SPE column with 3 mL MeOH followed by 3 mL H₂O. Transfer remaining aqueous from P/P tube to a (conditioned) SPE system consisting of a C₁₈ SPE column on bottom, PRS SPE column on top of the C₁₈, with a 70 mL reservoir atop the PRS; all on a vacuum manifold (allow to flow through at about 1 drop/sec). When level just reaches the top of PRS column, add 1 mL H₂O to columns and allow the PRS column to run dry into the C₁₈ column. Discard PRS column. When the level is even with the top of the C₁₈column, add 1mL 2% MeOH in H₂O and allow to run dry (under strong vacuum for a min). Elute the C₁₈ SPE with 4 mL MeOH into 15mL disposable P/P centrifuge tube. Evaporate MeOH eluate to dryness with the aid of acetonitrile in N-Evap with water-bath set at 50°C. The dried extracts are reconstituted into 250 µL of 0.1% formic acid, and filtered for injection into LC-MS system.
   
   
4. Instrument Operating Parameters
   
   Regardless of the instrument used, certain performance verification criteria should be incorporated into the operating parameters. These include mass calibration, tuning, and appropriate fragmentation patterns. Mass axis calibration should be performed according to the instrument manufacturers' specifications or according to internal laboratory MS standard operating procedures. Signal optimization (tuning) should be adjusted to maximize the abundance of ions of interest. Daily system suitability requirements (described in #7 of this section) should also be met. The following describes the specific operating procedures for the instrument used to validate this method in the developer's laboratory.
   
   
   1. Instrumental Configuration. LC/MS analysis is performed using a LCQ DECA mass spectrometer coupled to a TSP P4000 LC via an electrospray interface. The instrument is operated using positive and negative ion detection. The instrument was calibrated according to the manufacturer's instructions. The response for CAP was optimized by tuning on ion m/z 321. For tuning, CAP (1 ng/µL in mobile phase) was pumped through a syringe pump at 10 µL/min and then introduced into the LC flow (250 µL/min 80/20 0.1% formic acid/acetonitrile) via a T before entering the MS source. In the tune file the MS parameters were set to a prescan of 2 and a maximum inject time of 100 ms. The MS² parameters were also optimized using the tune function of the instrument. For this mode the prescan was set to 1 with a maximum inject time of 500 ms . The collision energy was optimized for both total MS² ion current, as well as for specific ions (m/z 194, 249) with no significant differences (optimal collision energy was 24-26% in all cases). The spray voltage can be set at -4kV to -5kV. The lower setting (-4kV) will minimize arcing from the capillary to the spray needle without any apparent loss of sensitivity.
      
      
   2. Monitored Response. Using the ion trap, MS² was performed on the molecular ions for each of the analytes according to the following program:
      
      Program: Fixed MS² Acquisition
      Isolation width was set to 2 amu for MS² transitions.
      
      Time Segment 1: 10-15 minutes CAP
      Scan Event 1: (-) MS scan from 300-350 amu
      Scan Event 2: (-) MS² m/z 321.2 (CE 24%) scan from 100-300 amu
      Scan Event 3: (-) MS² m/z 323.2 (CE 24%) scan from 100-300 amu
      
      A UV/Vis diode array detector was also utilized with a scan range of 190-800nm and channel A set to 270 nm (bandwidth 9 nm) and channel B set to 236 nm (bandwidth 9 nm).
      
      
   3. Specific Operating Conditions. The electrospray interface was operated with a temperature of 275°C. The sheath gas was nitrogen at approximately 35 psi; the auxiliary gas was also nitrogen at approximately 6 psi (optimized for CAP signal). The mobile phase was at flow of 250 µL/min and a column oven was not used. Automated injections of 75 µL were made using "push loop" type injection. The LC flow was diverted away from the mass spectrometer for the first ten minutes. The MS was on from 10-15 minutes and the LC flow diverted again. The chromatographic gradient is as follows:

      Time (minutes)	% Acetonitrile	% 0.1% Formic Acid
      0-5	2	98
      6-18	20	80
      20-22	90	10
      23-28	2	98

      
      

5. Procedures for Instrumental Analysis of Samples, Controls, and Standards
   
   Standards are to be run with each set of samples (at the beginning and end of a set of samples, and in the middle of the sequence if many samples are being analyzed). At least two positive controls, i.e. fortified matrix should be run along with any unknown sample extracts. A blank matrix sample (negative control) and a reagent blank should also be run along with any unknown sample extracts and must demonstrate the absence of CAP. The fortified matrix control samples must demonstrate the confirmation criteria in the Validation Section #2v. A solvent blank (mobile phase) should be run initially and after an external standard to ensure that there was no carryover.
   
   
6. Calculations
   
   For qualitative analysis, the important factor is to obtain information to determine if the data meet the confirmation criteria described in the Validation Section #2v. Ion chromatograms from the full MS (m/z corresponding to [M -H]^-) and from MS² (m/z 194 corresponding to [M- H-(NH₂COCCl₂H)]^- from both fragmentation of both m/z 321 and 323) can be shown along with the MS² spectra averaged across the chromatographic peaks. In addition, extracted ion chromatograms for several ions m/z 194, and 257 in the MS² spectra of 321, as well as, ions m/z 194 and the summation of ions 257 and 259 in the MS² spectra of 323 should be shown with the S/N calculated for each of these MS² transactions. As scan data are obtained, relative abundances of representative ions can be estimated from the appearance of the MS² spectra, or from tabulation data. Integration of ion chromatograms is not necessary.
   
   
7. System Suitability
   
   The instrument should meet calibration and tuning criteria as described above. In addition, for each day's analysis, a standard mixture should be analyzed initially to determine the performance qualifications, or system suitability of the instrument. The analytes need to elute at the correct retention time; within ± 5% of what was observed for standards previously (unless column or mobile phase have been changed) and within the time-dependent window if used. It may require one or two injections of standard for compounds to elute at correct retention time if instrument has not been used recently. In addition, the response for 75 µL injection of a 1 ppb standard for CAP should be > 200,000 counts for the 321==> 194 MS² transition.

Validation Information

1. Validation Data
   
   Figure 1 shows chromatograms for a 1 ppb crawfish fortified extract.
2. Parameters Evaluated
   
   
   1. Recovery: Fortified matrix samples were analyzed at 1 and 2 ppb. Both levels demonstrated appropriate responses and met confirmation criteria. Quantitative recovery was not performed.
   2. Reproducibility: A series of standard injections (75 µ L injection size) were analyzed using the following standards: At 1 ppb (3 ng on-column) the reproducibility of standard injections as measured by the CAP 321 to 194 transition was 16% (n=6), at 0.25 ng (750 pg on-column), 19.9% (n=5) and at 0.1 ppb (300 pg on-column) it was 40% (n=4).
   3. Specificity: This method meets the specificity guidelines for confirmation methods outlined by Sphon (11) and recently elaborated in CVM's draft guidance (12). During the course of this investigation, several lots of control crawfish were analyzed and there were no significant interfering peaks in any of the control tissue samples analyzed using the mass filters as described.

      11. J. Assoc. Off. Anal. Chem. 61, 1247 (1978)
      12. Center for Veterinary Medicine (2001) Guidance for Industry: Mass Spectrometry for Confirmation of the Identity of Animal Drug Residues

      
      
   4. Sensitivity: For CAP, the ion trap instrument was able to confirm approximately 300-500 pg of standards on-column and crawfish tissue fortified at 1.0 ppb was confirmed with a 75 uL injection volume (final extract volume of 250 µL).
   5. Accuracy, Proof of Recovery from Authentic Samples
      
      Using an ion trap instrument the following criteria must be met for qualitative confirmation:

      For chloramphenicol:

      1. Spectral criteria: The ion m/z 194 [M- H-(NH₂COCCl₂H)]^- must be observed in the MS² spectra from both parent ions (m/z 321 and 323), and should be a predominant peak in the mass range m/z 100-270. 2) In addition, the ions corresponding to [M-H-(HCOCl)]^- (m/z 257 and 259 in MS² spectra of 323) must be observed with an approximate relative abundance to the base peal m/z 194 as is observed in the external standards. Other peaks that may be observed in the MS² spectra, but are not required for confirmation, include m/z 249 [M-H-(2HCl)], m/z 176 [m/z 194 - (H₂O)]^-, and m/z 152. Any other predominant ions in the spectra should be able to be explained, i.e. present in reagent or matrix blank.
      2. Signal threshold (ST) criteria: a) The signal for the peak at the correct retention time for CAP in the ion chromatogram of 194 from the MS² spectra should be equal to or greater than ten times the negative control (blank matrix). b) The signal for the ions corresponding to [M-H-(HCOCl)]^- (m/z 257 from 321 and summation of m/z 257 and 259 from MS² of 323) should be equal to or greater than ten times the negative control (blank matrix).
      3. Retention time criteria: The retention time should be ±5% of external standards run on that day.

      
      
   6. Practicality, Sample Throughput, Solvents and Time Requirements: Extraction and LC/MS analysis of 6-8 samples can be accomplished in one day/overnight. For example, initial extraction can be performed in 5 hours. Each LC/MS run takes 28 minutes therefore 6 sample analyses (bracketed by analysis of standards, separated by solvent blanks) can be done in 8-12 hours.

Quality Control Points

1. Critical Points
   
   
   1. Extraction: When filtering, be careful that the syringe filter does not disengage.
      
      
   2. Chromatography: A formic acid/acetonitrile mobile phase at 0.25 mL/min on a semi-micro phenyl column resulted in the best chromatographic performance and electrospray sensitivity. The migration of peaks, especially at the beginning of the chromatographic analysis, can be a problem and several injections of standard may be necessary to allow compounds to "settle" into reproducible retention time. Retention times are stable during continuous sequences, even as long as 40-50 samples. It is important with this matrix to divert most of the LC effluent away from the MS (only sampling the 10-15 time interval) to prevent clogging of stainless steel capillary at the MS orifice and resulting instrument performance.
      
      
   3. Mass spectral analysis: In addition to obtaining good agreement between samples and standards analyzed on the same day, a review of the data shows that the relative abundances of ions obtained different days is also very reproducible.
      
      
2. Performance Specifications:
   Performance Specifications are outlined above in Procedures section #4.ii (tuning of mass spectrometer), #7 (system suitability for standards) and the Validation section #2.v (criteria for confirmation).
   
   
3. Stability:
   Stability of residues in crawfish stored for extended periods of time was not evaluated.
   
   
4. Safety:
   Standard laboratory safety practices (lab coats, eye protection) should be followed. In addition any safety precautions listed in the determinative SOP for preparation of reagents should be followed. Also follow instrument manufacturers guidelines for safe operation of electrospray LC/MS (particularly with respect to high voltages, high current, and high temperatures).

Figure 1. Extract from crawfish fortified with 1 ppb CAP.

Extracted ion chromatograms for MS² (A) m/z 194 from m/z 321 (B) m/z 257 from m/z 321(C) m/z 194 from m/z 323 and (D) m/z 257 +259 from m/z 323. (E) MS² spectrum for m/z 321 (F) MS² spectrum for m/z 323.