Online Postcolumn Derivatization

In this approach (Table 2) injection-separation steps are followed by online derivatization, using automated, fully online instrumentation and methods. This technique utilizes post-column reactors (low dead volume mixing tees, knitted open-tubular reactors, low dead volume reaction coils, etc.) in which the chemical regents are introduced to the LC eluent. A delay time is needed (reaction dependent) to convert the analyte to product(s), and the entire solution, along with excess reagent(s), is introduced into the detector. This approach also allows for online liquid-liquid extraction, ion suppression (dual column ion chromatography), pH adjustment, organic solvent addition, basic hydrolysis reactions, additional chemical reactions modifying the solutes prior to the derivatization step (e.g. oxidation of imidazole ring in proline and hydroxyproline for their assay by the OPA reaction), enzyme addition, and the use of post-column, immobilized reagents or enzymes. There are many chemical reactions that have been employed post-column online: sequential reactions, solid-phase/catalytic enhanced reactions (e.g. carbamate detection), microwave digestion of proteins, photochemical reactions, etc. However, there are severe constraints or requirements on the nature of the reagent solvent and solution that can be mixed with the LC effluent, detector transparency of such solvents, prevention of analyte derivative precipitation before detection, mixing of reagents with analyte, lack of mixing noise, need for additional instrumentation, mixing tees, connecting joints, and extra tubing connections. Nevertheless, at least in LC areas, this particular mode has been the most widely employed and applied.

Specific Recommendations for Successful Application of Derivatization in Liquid Chromatography

It is clear that there are numerous approaches to successful derivatization possible in various modes of LC, including reversed-phase, ion exchange, normal phase and hydrophobic interaction. There are perhaps too many choices as to which specific reagent will prove applicable for a new analyte, or how to best optimize and apply any given reagent, much less what might prove the optimal LC conditions for the final derivatives. A rational approach to derivatiz-ation for all LC is called for. Such rational designs for method development, optimization and validation in HPLC are available from the literature. A rational approach to developing, optimizing and then validating a derivatization method for LC is described below.

1. Know the structure of the analyte(s), what functional groups are present for tagging, and what types of reactions might be employed. A good knowledge of organic chemistry is needed and available at this stage. Some of the existing texts on derivatizations for HPLC should be utilized.

2. What are the requirements of the final derivatiz-ation-LC method? It is necessary to decide what detection limits are needed, what sample matrices will be analysed, what limits of quantitat-ion must be realized, what resolution (sample dependent) will be needed, and so forth.

3. What is known in the literature about the LC of the analyte of interest, as a standard pure compound (without regard to sample matrices yet)? Are conditions reported for underivatized analysis, and what conditions have been already described and optimized? Could these be eventually utilized for simple derivatives of the original analyte? What modifications might be needed to resolve the analyte derivatives? Are any tagging methods already reported for GC or thin-layer chromatography (TLC) that might prove applicable in LC? What types of reagents have been described? What were the specific reaction conditions already optimized for this derivatization scheme?

4. Perform simple, test tube reactions on a standard of the analyte offline, away from the LC instrument, to optimize reaction conditions and to demonstrate the nature of the products formed, their number, derivatization yield, ease of product work-up prior to LC, etc. Utilize TLC, gas chromatography (GC), LC, and whatever other analytical tools are available to determine which reagents will tag the analyte, the nature of the products formed. Follow the optimization steps described below.

5. Optimize the derivatization conditions in terms of the usual reaction parameters: time, solvent, pH, temperature, catalysts. This can be performed univariately or multivariately, even using computer algorithms (simplex/multiple routines) to realize surface maps of conditions leading to optimal formation of the desired derivative. Whatever the optimization routine used, the final conditions need to be compatible with pre- or post-column LC reaction requirements (instrumental, solvents, mixing). Optimize reaction conditions and demonstrate formation of the desired derivative before introduction into the LC instrument.

6. Demonstrate the formation of derivative, nature of the derivative (structure), purity of standard derivative, per cent derivatization (yield), etc., using standard organic chemistry methods (elemental analysis, mass spectrometry, Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy). What is the nature of the derivative obtained from the analyte? What is its exact structure, solubility, stability to various LC solvents, detection properties (UV, FL, EC), etc.?

7. How does the final derivatization approach change the possible ionization states of the original analyte? What modifications to the separation conditions of the original, untagged analyte must now be made to accommodate the nature of the derivatized species (e.g. ion exchange chromatography (IEC) changes)? Will the new tag(s) induce additional charges on the original analyte that will then affect LC mobility, migration times, resolutions, etc.? Will the tags induce unwanted hydrophobic properties to the tagged species affecting solubility, migration tendencies, resolution, efficiency and peak shape? How do we then accommodate such structural and physical/chemical property changes, how do we know what those changes really are before any LC methods development is pursued? Will the newly tagged species still permit host-guest complexa-tion, such as with cyclodextrins, crown ethers and other complexation additives to the LC buffer?

8. Now utilize the standard derivative to optimize the LC conditions, again consulting the literature to determine if this derivative or an analogous structure has already been described along with specific LC operating conditions. Utilize those conditions or slight variations to realize optimized LC conditions for your standard derivative. This may require optimization by univari-ate or multivariate methods, perhaps using computer algorithms, varying one parameter at a time to generate a surface map demonstrating optimized conditions. This is similar to resolution maps in LC via DryLab from LC Resources. There are other computer programs in the literature that might prove useful in this area of LC separation optimization for the standard derivative.

9. Demonstrate analytical figures of merit with standard derivative, based in part on original method/assay requirements, such as linearity of calibration plots possible (UV, FL, EC), detection limits, limit of quantitation, accuracy and precision of quantitations possible, robustness of the LC conditions to small operational changes (pH, temperature, solvent, ionic strength, voltage applied, sample introduction, etc.), time per analysis, cost per analysis, instrument/method preparation, etc. This is still all derived for standards of the derivative, and not yet with actual analyte or samples.

10. Demonstrate analytical figures of merit with standard analyte, exactly as above, but now introducing the actual derivatization steps required to convert the original standard analyte into the derivative.

11. Demonstrate analytical figures of merit with actual sample containing known levels of analyte, including all method requirements: limit of quan-

titation (LOQ), limit of detection (LOD), linearity of calibration plots, ruggedness, robustness, reproducibility, repeatability, accuracy/ precision of quantitations, time per analysis, cost per analysis and sample preparation requirements.

12. Validate final, optimized method with known samples containing known levels of analyte using double-blind spiking, standard reference materials (if available), comparison with currently accepted method on split, spiked samples (known levels), and finally interlaboratory collaborative studies. Assemble all final data in terms of accuracy and precision, reproducibility from laboratory to laboratory, repeatability within one laboratory, ruggedness from laboratory to laboratory, robustness within any given laboratory, all in terms of qualitative identification of analyte present, and then final quantitative information in terms of accuracy and precision of such measurements.

13. Write up final procedure and protocols for performing the final, overall derivatization-LC method, including the necessary sample preparation steps, isolation of analyte from matrix (if required) before derivatization, possible derivat-ization of analyte in sample matrix followed by isolation of derivative, or derivatization of analyte in sample matrix with direct injection of crude mixture into LC with minimal (if any) sample preparation (dilute/shoot). Include all possible procedures and reagents, chemicals, solvents and instrumentation needed for another laboratory to reproduce, repeat, and obtain valid results using the newer method in their hands/ laboratories.

14. Distribute the final protocols and procedures to all those laboratories that participated in the interlaboratory collaborative studies, so that they can validate and demonstrate reproducibility of the overall optimized methods involving derivat-ization-LC operations and conditions.

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