Autosamplers calibration

This example presents four analytical situations (a) good control, (b) sudden shift in accuracy - perhaps the calibration material has become contaminated, (c) a gradual shift in accuracy-perhaps a reagent has exceeded its expiry date, (d) very poor precision - perhaps an autosampler is working erratically. 

HPLC column, Merck, LiChrospher 60 RP Select B, 5 itm, 150 x 4-mm i.d. Autosampler vials, 1.8-mL Assorted laboratory glassware Autopipettes, calibrated... 

Prepare a calibration curve in the following manner. Measure 1 mL of control water into a series of 1.8-mL autosampler vials. Fortify each water sample with 200 o.L of the appropriate calibration standard to make a 0.1, 0.5 and 2.5ngmL calibration curve. Mix the sample by vortexing or shaking the vial and analyze 200 iL by LC/ESI-MS/MS. For well and lysimeter water, mix 1 mL of sample water with 200 uL of the lOngmL" mixed internal standard solution in a 1.8-mL autosampler vial, cap the vial, mix the contents and analyze 200 iL by LC/ESIMS/MS. 

This chapter reviews the principles and strategies used for HPLC system calibration that includes the pump, the detector, the autosampler, and the column oven. A case study is used to illustrate the development of the calibration procedures for all system modules and the rationale of setting up acceptance criteria that balance productivity and compliance. 

The calibration order is detector => pump => autosampler (as the detector is often used to calibrate other modules). 

FIGURE 10 Part of the MS Word template form to document the calibration of the autosampler. A 4-page template was used to document the results obtained for HPLC calibration so any laboratory notebook entries are not needed. Reprinted with permission from Reference 6. 

Postpreparative Stability. The stability of processed samples, including the resident time in the autosampler, should be determined. The stability of the drug and the internal standard should be assessed over the anticipated run time for the batch size in validation samples by determining concentrations on the basis of original calibration standards. Reinjection reproducibility should be evaluated to determine if an analytical run could be reanalyzed in the case of instrument failure. 

Transfer 100 pi of the clear urine, calibration standard or control into an autosampler vial (Gilson). 

Calibration is time consuming when performed correctly. It may require 1 or 2 days to perform all the necessary steps (i.e., prepare stocks, filter, measure absorbance, check purity, dilute, mix, and inject calibrants). Once the stock solutions and mixed calibration solutions have been prepared, a calibration check can be performed in -4 hr. Sample preparation, depending on the matrix, may require a few minutes or a few hours. If an autosampler is unavailable for overnight injection the extracts are typically stable overnight, refrigerated at - 20° to 4°C. It is prudent to maintain the autosampler tray temperature from 4° to 15°C to reduce sample degradation. HPLC analysis of the extracted sample requires 20 to 60 min. Typically one technician can extract 12 to 24 samples per day to be analyzed overnight or the next day. 

Samples from the 40-mL VOA vials were split for chromate and PCE analyses and typically analyzed within 48 hours of collection. Chromate concentration was determined via an HPLC method using a Gilson Model 116 UV detector set at 365 nm and a 2- by 150-mm Waters Nova-Pak C18 60A HPLC column packed with 4-pm particles. The mobile phase consisted of 5-mM tert-butylammonium hydrogen sulfate buffered to pH 4.4 with NaOH with 10% acetonitrile (v/v) as a modifier. The eluent flow rate was 0.8 mL min-i Samples were filtered through a0.45-pm filter as they were injected by an Alcott 708 autosampler with a 0.1-mL sample loop. The typical run time was 4 min with a calibration range of 0.05 to 20 mg L 1 (0.001 - 0.38mmol L 1) Cr as chromate. 

The ICP was a Perkin-Elmer 3000DV with an AS90 Autosampler, which has an instrument detection limit of about 1 ppb (for most elements) with a linear calibration up to 100 ppm (for most elements). Solid samples were prepared via microwave digestion in concentrated nitric and hydrochloric acids, then diluted to volume. The ICP was calibrated and verified with two independent, certified standard sets. Spikes and dilutions were done for each batch of samples to check for and/or mitigate any matrix effects. The ICP process ran a constant pump rate of 1.5 mL/min for all samples and standards during analysis. A 3 mL/min rinse and initial sample flush were used to switch between each sample and standard. The plasma was run at 1450 W with argon flow. Trace metal-grade (sub-ppb) acids and two independently NIST-certified calibration standard sets were used for calibration and method verification. 

Injectors After dissolution in mobile phase or other suitable solution, compounds to be chromatographed are injected into the mobile phase, either manually by syringe or loop injectors, or automatically by autosamplers. The latter consist of a carousel or rack to hold sample vials with tops that have a pierceable septum or stopper and an injection device to transfer sample from the vials to a calibrated, fixed-volume loop from which it is loaded into the chromatograph. Some autosamplers can be programmed to control sample volume, the number of injections and loop rinse cycles, the interval between injections, and other operating variables. 

Reliable quantitative results are obtained by external calibration if automatic injectors or autosamplers are used. This method involves direct comparison of the peak responses obtained by separately chromatographing the test and reference standard solutions. If syringe injection, which is irrepro-ducible at the high pressures involved, must be used, better quantitative results are obtained by the internal calibration procedure where a known amount of a noninterfering compound, the internal standard, is added to the test and reference standard solutions, and the ratios of peak responses of the analyte and internal standard are compared. 

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