Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses

Therapeutic drug monitoring of antibiotics is increasingly recognized as an important element for the implementation of national and local antibiotic stewardship programs. To meet this increasing demand of antibiotic therapeutic drug monitoring in medical facilities, we present a protocol for the quantification of antibiotics in circulation that aims to adjust the dosage to the susceptibility of the involved pathogens. The presented mass spectrometry-based protocol was optimized for the quantification of the most commonly used antibiotics in intensive care units, namely cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin.

Due to the broad calibration range, the method can also be used to calculate the pharmacokinetic area under the curve for an administered antibiotic. With a total turnaround time of approximately 30 minutes, the presented protocol is suitable for daily antibiotic therapeutic drug monitoring in clinical laboratories. First spike nine volumes of drug-free serum with one volume of previously prepared 10-fold concentrated spike solutions to obtain the serum calibrators zero through seven and quality controls A through D.For example, add 0.5 milliliters of spike solution to 4.5 milliliters of serum in a 10-milliliter polypropylene tube and incubate it for 15 minutes at four degrees Celsius on a roller mixer at 50 rpm.

Use a repetitive pipette to generate 100-microliter aliquots of the calibrators and quality controls in 1.5-milliliter polypropylene tubes. Store the calibrators, quality controls, and previously prepared antibiotic stock solutions at minus 80 degrees Celsius for up to six months. After preparing six internal standard stock solutions, combine them in a 1.5-milliliter polypropylene tube to yield a five-fold concentrated internal standard mix.

Add the desired internal standards to 965 microliters of 25%methanol in water. Store the internal standard stock solutions and the five-fold concentrated internal standard mix at minus 80 degrees Celsius. To prepare mobile phase A, add approximately 500 milliliters of HPLC grade water to a 1, 000-milliliter volumetric flask.

Add one milliliter of formic acid, 10 milliliters of one-molar ammonium formate, and fill the flask to 1, 000 milliliters with HPLC grade water. Then transfer the mobile phase solution to a clean glass bottle. To prepare mobile phase B, add HPLC grade absolute methanol to a clean glass bottle.

To prepare the purge solvent, add approximately 500 milliliters of distilled water to a 1, 000-milliliter volumetric flask. Add 70 milliliters of absolute methanol, one milliliter of formic acid, and fill the flask to 1, 000 milliliters with distilled water. Transfer the solvent to a clean glass bottle.

Connect the bottles with mobile phases A and B and the purge solvent to the HPLC system. Make sure that the needle wash solvent tube is connected to the bottle containing mobile phase B.To prepare the precipitation agent, add 2.5 milliliters of methyl tert-butyl ether to a 25-milliliter volumetric flask and fill to 25 milliliters with absolute methanol. Place a C8 reversed phase column into the column chamber, then connect the column to the HPLC and mass spectrometer in the direction of the flow.

In order to generate the sample list, open the corresponding sample measurement master file template and add the patient samples to be processed. Generate groups of up to 20 patient samples and flank them with the corresponding quality control pair. Using the Inlet file control software, set the Wet Prime function to 50%mobile phase A and 50%mobile phase B and wet prime for two minutes with a flow rate of one milliliter per minute.

Next, refresh the syringe by executing six strokes of 600 microliters in the control software. To equilibrate the C8 reversed phase column, turn on the the flow in the inlet file and flush it with 7%mobile phase B and 93%mobile phase A for a minimum of five minutes, using a flow rate of 0.4 or 0.5 milliliters per minute. Visually verify the column temperature of 30 degrees Celsius.

With a repetitive pipette, add 25 microliters of the internal standard mix to the thawed calibrator, QC sample, or patient serum in a 1.5-milliliter polypropylene tube and vortex the tube for a few seconds. Then incubate the mixture for five minutes at room temperature on a benchtop shaker. After incubation, add 150 microliters of the precipitation reagent to the sample internal standard mix with a repetitive pipette.

Vortex the tube for a few seconds and then incubate for five minutes at room temperature on a benchtop shaker. Following this, centrifuge the suspension at 20, 000 times gravity in a tabletop centrifuge for 10 minutes at four degrees Celsius. Dilute the supernatant one in three with HPLC grade water in a glass vial with a micro insert.

Then, place the sample in the HPLC autosampler. Manually start the HPLC-MS/MS analysis in the sample measurement control file. To process the samples, open the corresponding sample measurement control file, select the calibrators, quality controls, and patient samples and evaluate them with the antibiotics quantification method.

Check whether the peaks for a specific analyte are properly integrated. Inspect the peaks for each calibrator, QC, and patient sample and manually reintegrate them at the baseline if necessary. Study the calibration curve and examine whether it fulfills linearity over the entire calibration range, a calibration coefficient greater than 0.99, a deviation of each calibration standard within plus or minus 15%of the nominal value except for the lower limit of quantification where a plus or minus 20%is required.

Reject a calibration standard not complying with the above-mentioned criteria and reevaluate the calibration curve, including the regression analysis. Now, study the quality controls and examine whether the deviations are within plus or minus 15%of the nominal value. If the quality controls for a specific batch do not fulfill the quality criteria, the samples must be reinjected or even be reprocessed if significant deviations persist.

Measured concentrations of a batch must also be evaluated whether they are plausible or not. If the concentration of a patient's sample exceeds the concentration of the highest calibrator, dilute the sample with distilled water up to one in five before the sample cleanup. Repeat the previous cleanup steps for that specific sample and reprocess it.

The calculated retention factors for all analytes were 2.8 to 4.2. A sample chart list for the processed samples is shown here. The key parameter is the response gradually increasing with the analyte concentration due to the constant amount of added isotope-labeled internal standard.

The calibration curve and quality controls for the example shown here fulfill all quality criteria. The coefficient of determination r-squared is greater than 0.995 and the deviation of the calibrators and the QC samples is within plus or minus 15%of the nominal value. The measured parent-to-daughter ion transitions show four peaks at the same retention time.

The two upper peaks depict two transitions that are measured for the analyte of interest and the lower two peaks represent the transitions for the corresponding isotope-labeled internal standard. The first patient has a high serum trough level of 83.4 milligrams per liter of piperacillin that is also sufficient for problem pathogens. The second patient has a concentration of approximately 0.2 milligrams per liter, which is below the lowest calibrator.

The third patient has low piperacillin trough concentration of only 5.3 milligrams per liter that is not sufficient for the majority of pathogens. The presented protocol is suitable for isotope dilution HPLC-tandem mass spectrometry-based quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin in human serum. The method uses a rather simple instrument setup and will be there transferable to various mass spectrometry-based platforms.

Chromatographic separation of the analytes of interest can also be obtained with a flow rate of 0.4 milliliters per minute as given in the present video. The described method can be used for pharmacokinetic studies as the calibration range allows both the quantification of concentrations close to the minimal inhibitory concentration of a susceptible pathogen as well as peak concentrations that are obtained with bolus administration regimens. After its development this method has been implemented in a mass spectrometry-based care facility for the antibiotic therapeutic drug monitoring in critically ill patients.

Due to the fact that beta-lactam antibiotics are chemically unstable once they are dissolved, the quality of the pre-analytical phase such as sample storage plays a vital role in obtaining reliable quantification results. Don't forget that working with organic solvents is hazardous and precautions such as working in a fume should always be taken while preparing chromatography solvents and the precipitation reagent.