1. (1) CMTLs must create and maintain Retention Time Windows for each Target Analyte, Surrogate, and Internal Standard for each Testing Field in which chromatography is used for analysis.

   1. (a) The Retention Time Window is calculated by multiplying three (3) times the standard deviation of the Retention Times of no less than seven (7) injections of a standard containing the same concentration of the Target Analyte. The result is then added to and subtracted by the expected Retention Time to create the Retention Time Window. The Retention Time Window cannot be less than the expected Retention Time of the peak plus or minus 0.03 minutes. The expected Retention Time must be updated at the time of Calibration. If a Calibration is not performed, the CMTL must update the expected Retention Time when a shift in Retention Times is observed at the beginning of each analytical run using the first injection of a CCV standard.
   2. (b) Identification of a Target Analyte peak outside of its established Retention Time Window is treated as a Manual Integration as described in subsection (5). A CMTL may choose to not allow identification outside of a Retention Time Window as part of the SOP required by subsection (6).

2. (2) The resolution between a Target Analyte’s chromatographic peak and an adjacent peak, whether an interferent peak or a preceding or succeeding Target Analyte, must be greater than or equal to 1.0 using the equation:

   1. (a) This calculation must be performed under the following circumstances:

      1. During validation of the instrument method for any Target Analyte whose chromatographic peak and an adjacent peak, whether an interferent peak or a preceding or succeeding Target Analyte does not achieve Baseline Resolution.

      2. During review of analytical data in which the resolution between any Target Analyte whose chromatographic peak and an adjacent peak does not achieve Baseline Resolution.

      3. During review of analytical data in which the resolution between a Target Analyte and an adjacent peak that has shifted Retention Times and appears to not have a resolution greater or equal to 1.0.

   2. (b) If upon calculation, the resolution is less than 1.0, the CMTL must discontinue conducting the analysis until the CMTL is able to achieve a resolution of greater than or equal to 1.0. Target Analytes with a resolution from adjacent peaks less than 1.0 is not reportable.

   
   

   where R is the resolution, RT is the Retention Time of the peaks, and W is the width of the peaks at one half the height of the peaks. For Gas Chromatography and Liquid Chromatography resolution is required for Selective Ion Monitoring (SIM) and Multiple Reaction Monitoring (MRM) windows not for the overall responses.

3. (3) Integration parameters for Automatic Integration must be set at time of Calibration and not changed until the next Calibration.
4. (4) All peaks that meet criteria identified in the CMTL’s Quality Assurance Manual and SOPs, which include but are not limited to Retention Time, Signal-to-noise Ratio, and ion ratios, must be integrated and uploaded to the CMTL’s tracking system to determine if the instrument response equates to a concentration of greater than the LOD.

5. (5) CMTLs may perform Manual Integrations when the Automatic Integration performed by the chromatographic software is in error.

   1. (a) CMTLs must maintain the original Automatic Integration performed by the chromatographic software and the final Manual Integration performed by the Analyst as part of the Data Package in Rule 64-4.311, F.A.C.
   2. (b) CMTLs must utilize the audit trail functionality of all chromatographic software. This functionality must also be used in the annual review required by subsection (8).
   3. (c) All Manual Integrations must have a record of the Analyst who performed them, the date they were performed, and the reason for manually integrating the peak.
   4. (d) Raw data resulting from a manually integrated peak must be automatically identified by the chromatographic software.
   5. (e) All Manual Integrations must be reviewed by a second Analyst before reporting the data and have a record of the name of the reviewer and date of the review. The reviewer must be an Analyst trained on the CMTL’s Manual Integration policies and have a valid IDOC for the Testing Field being reviewed.
   6. (f) Manual Integrations must never be used to force a Calibration or Quality Control Sample to pass method and/or regulatory criteria. Manual integrations must not be used to force a client sample to calculate below the Acceptable Limit. Manual integrations may not be performed where there are no errors present in either the Automatic Integration performed by the chromatographic software or a previous Manual Integration. This includes, but is not limited to, Peak Shaving and Peak Enhancement.
   7. (g) All integrations, whether automatic, manual, or both, across all Calibration Standards, calibration verification standards, batch Quality Control Samples, and client samples must be consistently integrated.
   8. (h) A CMTL may not use Manual Integrations to replace proper instrument maintenance or other corrective or preventative actions to improve chromatographic performance.
   9. (i) All automatically integrated and manually integrated peaks must meet a Signal-to-noise Ratio of at least three (3) in order to be reported.

6. (6) CMTLs must create, maintain, and follow an SOP for chromatography and Manual Integration standards. The SOP must include but is not limited to the following topics:

   1. (a) A CMTL must not establish a policy prohibiting Manual Integrations;
   2. (b) All requirements contained in subsections (1) through (5);
   3. (c) Definition of Peak Shaving and an explanation of its prohibition;
   4. (d) Definition of Peak Enhancement and an explanation of its prohibition;
   5. (e) Definition of consistent integrations and how to perform them in difficult Matrices, including interference peaks, fronting, tailing, and fluctuating baselines; and
   6. (f) Excessive Manual Integrations on a single peak and proper corrective measures.
7. (7) CMTLs must have annual training on Manual Integration policies and procedures for all Analysts that perform analyses in a Testing Field utilizing chromatography. All Analysts must be trained in the CMTL’s Manual Integration policies and procedures as part of their IDOC in a Testing Field utilizing chromatography. This annual training should include but is not limited to all topics in subsection (6).

8. (8) The Laboratory Director, or their designee, must perform an annual review of at least three (3) analytical runs of each Testing Field utilizing chromatography and at least three (3) analytical runs for each Analyst across all Testing Fields in which the Analyst performs analyses utilizing chromatography.

   1. (a) The Laboratory Director, or their designee, must not be the original Analyst or the original reviewer of the data as required in paragraph (5)(e).

   2. (b) A report must be generated after the review is completed and be sent to the department at OMMULabs@flhealth.gov within five (5) business days of completion. The report must contain:

      1. The CMTL’s name and address;

      2. The Testing Field that was reviewed;

      3. The Analyst that was reviewed;

      4. A copy of all Automatic Integrations and Manual Integrations reviewed;

      5. A narrative of all observations and conclusions based on the review;

      6. Any corrective action plans as a result of the review; and

      7. The name and signature of the Laboratory Director, or their designee, who performed the review.

Rulemaking Authority Art. X, § 29, Fla. Const., 381.988(3), 381.988(9) FS. Law Implemented Art. X, § 29, Fla. Const., 381.988 FS. History–New 5-28-26.