Name: multiplex therapeutic drug monitoring by isotope-dilution hplc-ms/ms of antibiotics in critical illnesses
Uploaded: 2018-08-30T15:00:00
Description: Watch this Scientific Journal Video about Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses at JoVE.com

The authors thank Dr. Sch&#252;tze for his help with establishing the presented method and Dr. Zoller for the valuable input regarding the proper calibration range. The authors also acknowledge the technical staff of the mass spectrometry facility.

NOTE: It is recommended to work in a fume hood when handling organic solvent, such as methanol. Prepare all buffers and mobile phases in volumetric flasks. If not otherwise specified, the solutions can be stored at room temperature for up to 1 month after preparation.
1. Preparation of the Calibrators and Quality Control Samples
NOTE: A corresponding data analysis sheet for the preparation of stock and spike solutions is given in the Supplemental File...

<ol>
	<li>Fleischmann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Global+Incidence+and+Mortality+of+Hospital-treated+Sepsis.+Current+Estimates+and+Limitations.">Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations.</a> American Journal of Respiratory and Critical Care Medicine. 193 (3), 259-272 (2016).</li><li>Dellinger, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+Sepsis+Campaign:+international+guidelines+for+management+of+severe+sepsis+and+septic+shock,+2012.">Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012.</a> Intensive Care Medicine. 39 (2), 165-228 (2013).</li><li>Lodise, T. P., Drusano, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+pharmacodynamics:+optimal+antimicrobial+therapy+in+the+intensive+care+unit.">Pharmacokinetics and pharmacodynamics: optimal antimicrobial therapy in the intensive care unit.</a> Critical Care Clinics. 27 (1), 1-18 (2011).</li><li>Macedo, R. S., Onita, J. H., Wille, M. P., Furtado, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+pharmacodynamics+of+antimicrobial+drugs+in+intensive+care+unit+patients.">Pharmacokinetics and pharmacodynamics of antimicrobial drugs in intensive care unit patients.</a> Shock. 39, 24-28 (2013).</li><li>Petersson, J., Giske, C. G., Eliasson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+dosing+of+piperacillin+and+meropenem+fail+to+achieve+adequate+plasma+concentrations+in+ICU+patients.">Standard dosing of piperacillin and meropenem fail to achieve adequate plasma concentrations in ICU patients.</a> Acta Anaesthesiologica Scandinavica. 60 (10), 1425-1436 (2016).</li><li>Abdul-Aziz, M. H., Lipman, J., Mouton, J. W., Hope, W. W., Roberts, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+pharmacokinetic/pharmacodynamic+principles+in+critically+ill+patients:+optimizing+efficacy+and+reducing+resistance+development.">Applying pharmacokinetic/pharmacodynamic principles in critically ill patients: optimizing efficacy and reducing resistance development.</a> Seminars in Respiratory and Critical Care Medicine. 36 (1), 136-153 (2015).</li><li>Imani, S., Buscher, H., Marriott, D., Gentili, S., Sandaradura, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Too+much+of+a+good+thing:+a+retrospective+study+of+beta-lactam+concentration-toxicity+relationships.">Too much of a good thing: a retrospective study of beta-lactam concentration-toxicity relationships.</a> Journal of Antimicrobial Chemotherapy. 72 (10), 2891-2897 (2017).</li><li>Ventola, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibiotic+resistance+crisis:+part+1:+causes+and+threats.">The antibiotic resistance crisis: part 1: causes and threats.</a> Pharmacy &amp; Therapeutics. 40 (4), 277-283 (2015).</li><li>Pulcini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+stewardship:+update+and+perspectives.">Antibiotic stewardship: update and perspectives.</a> Clinical Microbiology and Infection. 23 (11), 791-792 (2017).</li><li>Cairns, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+a+multidisciplinary+antimicrobial+stewardship+team+on+the+timeliness+of+antimicrobial+therapy+in+patients+with+positive+blood+cultures:+a+randomized+controlled+trial.">The impact of a multidisciplinary antimicrobial stewardship team on the timeliness of antimicrobial therapy in patients with positive blood cultures: a randomized controlled trial.</a> Journal of Antimicrobial Chemotherapy. 71 (11), 3276-3283 (2016).</li><li>Baur, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+antibiotic+stewardship+on+the+incidence+of+infection+and+colonisation+with+antibiotic-resistant+bacteria+and+Clostridium+difficile+infection:+a+systematic+review+and+meta-analysis.">Effect of antibiotic stewardship on the incidence of infection and colonisation with antibiotic-resistant bacteria and Clostridium difficile infection: a systematic review and meta-analysis.</a> Lancet Infectious Diseases. 17 (9), 990-1001 (2017).</li><li>Roberts, J. A., Norris, R., Paterson, D. L., Martin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+drug+monitoring+of+antimicrobials.">Therapeutic drug monitoring of antimicrobials.</a> British Journal of Clinical Pharmacology. 73 (1), 27-36 (2012).</li><li>Paal, M., Zoller, M., Schuster, C., Vogeser, M., Schutze, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantification+of+cefepime,+meropenem,+ciprofloxacin,+moxifloxacin,+linezolid+and+piperacillin+in+human+serum+using+an+isotope-dilution+HPLC-MS/MS+method.">Simultaneous quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid and piperacillin in human serum using an isotope-dilution HPLC-MS/MS method.</a> Journal of Pharmaceutical and Biomedical Analysis. 152, 102-110 (2018).</li><li>Vogeser, M., Seger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitfalls+associated+with+the+use+of+liquid+chromatography-tandem+mass+spectrometry+in+the+clinical+laboratory.">Pitfalls associated with the use of liquid chromatography-tandem mass spectrometry in the clinical laboratory.</a> Clinical Chemistry. 56 (8), 1234-1244 (2010).</li><li>United States Pharmacopeia and National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+&#60;621&#62;.">Chapter &#60;621&#62;.</a> CHROMATOGRAPHY (USP 37-NF 32 S1). , 6376-6385 (2014).</li><li>Wong, G., Sime, F. B., Lipman, J., Roberts, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+we+use+therapeutic+drug+monitoring+to+improve+outcomes+from+severe+infections+in+critically+ill+patients?.">How do we use therapeutic drug monitoring to improve outcomes from severe infections in critically ill patients?.</a> BMC Infectious Diseases. 14, 288 (2014).</li><li>. Cefepime hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/050679s040lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/050679s040lbl.pdf</a> (2016)</li><li>. Meropenem: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2008/050706s022lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2008/050706s022lbl.pdf</a> (2006)</li><li>. Ciprofloxacin hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/019537s086lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/019537s086lbl.pdf</a> (2016)</li><li>. Moxifloxacin hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2010/021277s038lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2010/021277s038lbl.pdf</a> (2010)</li><li>. Linezolid: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2012/021130s028lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2012/021130s028lbl.pdf</a> (2011)</li><li>. Piperacillin and Tazobactam: Highlighs of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2017/050684s88s89s90_050750s37s38s39lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2017/050684s88s89s90_050750s37s38s39lbl.pdf</a> (2017)</li><li>Zander, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+biobanking+conditions+on+six+antibiotic+substances+in+human+serum+assessed+by+a+novel+evaluation+protocol.">Effects of biobanking conditions on six antibiotic substances in human serum assessed by a novel evaluation protocol.</a> Clinical Chemistry and Laboratory. 54 (2), 265-274 (2016).</li><li>Zander, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+piperacillin,+tazobactam,+cefepime,+meropenem,+ciprofloxacin+and+linezolid+in+serum+using+an+isotope+dilution+UHPLC-MS/MS+method+with+semi-automated+sample+preparation.">Quantification of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in serum using an isotope dilution UHPLC-MS/MS method with semi-automated sample preparation.</a> Clinical Chemistry and Laboratory. 53 (5), 781-791 (2015).</li></ol>

In this manuscript, we report the protocol for a simple and robust tandem mass spectrometry-based method for the quantification of frequently used antibiotics in ICU19, namely cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin14. A spreadsheet accompanies the manuscript for the preparation of antibiotic stock solutions, calibrators, and quality controls, taking into account the purity of the antibiotics and the molecular weight of their counterion...

Although antibiotics have revolutionized the practice of medicine, severe bacterial infections remain a leading cause of morbidity and mortality in critical illnesses1. In this regard, the prompt administration of a suitable anti-infective in an adequate dosage is of the uppermost importance for disease control2.
A growing body of evidence demonstrates that the empirical treatment with broad-spectrum antibiotics is becoming increasingly problematic with the complexity of patient populations. This is especially true for intensive care units (ICU), where a tremendous inter-individual variability of key pharmacokinetic (PK) parameters is frequently observed3,4. Accordingly, ICU patients are at imminent risk of sub-therapeutic levels with the danger of an insufficient therapeutic success5,6. Then again, patients are unnecessarily exposed to excessively high antibiotic concentrations that may result in serious adverse events with no clinical benefits7. Both the antibiotic misuse and the insufficient dosing have also fueled the dissemination of antibiotic resistance, which is becoming an ever-growing threat to public health8.
To improve the use of antibiotics and to preserve their effectivenessas long as possible, the World Health Organization has launched a global action plan on antimicrobial resistance in 20159. Antibiotic stewardship programs constitute an essential cornerstone of prudent antimicrobial use in national public health strategies10, helping clinicians to improve the quality of patient care11 and, at the same time, significantly reducing the antibiotic resistance12. Antimicrobial dosing in individual patients through the application of therapeutic drug monitoring (TDM) is a key instrument in this context13.
To date, commercially available TDM assays are only available for the glycopeptide antibiotics and aminoglycosides. The quantification of substances from other classes commonly requires an in-house method development or validation that can be cumbersome. We, therefore, present in detail the protocol for a robust mass spectrometry-based assay that can be used for the quantification of the most relevant antibiotics in ICU within their clinical relevant concentration ranges14. The method was recently established in our mass spectrometry facility and has been applied for the routine TDM in ICU since then. The procedure uses a straightforward and simple analytical setting with a uniform sample cleanup, allowing for the rapid implementation of antibiotic TDM in many facilities with mass spectrometry capabilities.
The protocol described here was optimized for the quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin in human serum, using isotope dilution liquid chromatography (LC) in combination with a tandem mass spectrometry (MS/MS). For the isotope dilution LC-MS/MS methodology, stable isotope-labeled compounds are added to a sample of interest with a specific matrix (e.g., serum). Isotope-labeled standards can be distinguished from their unlabeled counterpart, namely the analyte of interest, due to different molecular weights of the natural molecule and their fragmentation products, termed a parent-ion-to-daughter-ion transition. As isotope-labeled compounds have an almost identical overall physicochemical behavior compared to their unlabeled counterpart, they are ideal internal standards for the MS/MS, allowing a nearly matrix-independent analyte quantification with a high degree of accuracy15. Nowadays, many stable isotope-labeled internal standards that can be used for small-molecule quantification, including the TDM of antimicrobials, are commercially available.
The chromatographic separation of the antibiotic analytes in the described protocol is performed with an analytical C8 alkyl-chain-length reverse-phase column (100 mm x 2.1 mm, 3 &#181;m particle-size). During the method development, the internal standard normalized matrix factors for all analytes was between 94.6% and 105.4%, with a coefficient of variation of &#8804;8.3%14.

<table>
	<tbody>
		<tr>
			<td>cefepime hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>1097636</td>
			<td>USP Reference Standard</td>
		</tr>
		<tr>
			<td>meropenem trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>Y0001252</td>
			<td>EP Reference Standard</td>
		</tr>
		<tr>
			<td>ciprofloxacin</td>
			<td>Sigma-Aldrich</td>
			<td>17850</td>
			<td></td>
		</tr>
		<tr>
			<td>moxifloxacin hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>SML1581</td>
			<td></td>
		</tr>
		<tr>
			<td>linezolid</td>
			<td>Toronto Research Chemicals</td>
			<td>L466500</td>
			<td></td>
		</tr>
		<tr>
			<td>piperacillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>93129</td>
			<td></td>
		</tr>
		<tr>
			<td>cefepime-13C12D3 sulfate</td>
			<td>Alsachim</td>
			<td>C1297</td>
			<td>Isotope labelled internal standard for cefepime</td>
		</tr>
		<tr>
			<td>meropenem-D6</td>
			<td>Toronto Research Chemicals</td>
			<td>M225617</td>
			<td>Isotope labelled internal standard for meropenem</td>
		</tr>
		<tr>
			<td>ciprofloxacin-D8</td>
			<td>Toronto Research Chemicals</td>
			<td>C482501</td>
			<td>Isotope labelled internal standard for ciprofloxacin</td>
		</tr>
		<tr>
			<td>moxifloxacin-13C1D3 hydrochloride</td>
			<td>Toronto Research Chemicals</td>
			<td>M745003</td>
			<td>Isotope labelled internal standard for moxifloxacin</td>
		</tr>
		<tr>
			<td>linezolid-D3</td>
			<td>Toronto Research Chemicals</td>
			<td>L466502</td>
			<td>Isotope labelled internal standard for linezolid</td>
		</tr>
		<tr>
			<td>piperacillin-D5</td>
			<td>Toronto Research Chemicals</td>
			<td>P479952</td>
			<td>Isotope labelled internal standard for piperacillin</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>JT Baker</td>
			<td>8402</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>JT Baker</td>
			<td>4218</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid</td>
			<td>Biosolve</td>
			<td>6914132</td>
			<td></td>
		</tr>
		<tr>
			<td>acetic acid</td>
			<td>Biosolve</td>
			<td>1070501</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium formate</td>
			<td>Sigma-Aldrich</td>
			<td>70221-25G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Butyl methyl ether</td>
			<td>Merck</td>
			<td>101845</td>
			<td></td>
		</tr>
		<tr>
			<td>Fortis 3 &#956;m C8 100 * 2.1 mm</td>
			<td>Fortis</td>
			<td>F08-020503</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti-PEEK-encased Prifilter (2 &#956;m)</td>
			<td>Chromsystems</td>
			<td>15011</td>
			<td></td>
		</tr>
		<tr>
			<td>2795 Alliance HPLC system</td>
			<td>Waters</td>
			<td>176000491</td>
			<td></td>
		</tr>
		<tr>
			<td>Quattro micro API Tandem Quadrupole System</td>
			<td>Waters</td>
			<td>720000338</td>
			<td></td>
		</tr>
		<tr>
			<td>QuanLynx 4.1 software</td>
			<td>Waters</td>
			<td>/</td>
			<td>Data evaluation software provided by the mass spectrometer manufacturer</td>
		</tr>
	</tbody>
</table>

multiplex therapeutic drug monitoring by isotope-dilution hplc-ms/ms of antibiotics in critical illnesses

There is an ever-increasing demand for the therapeutic drug monitoring of antibiotics in many clinical facilities, particularly with regard to the implementation of hospital antibiotic stewardship programs.
In the current work, we present a multiplex high-performance liquid chromatography-tandem mass spectrometry (HPCL-MS/MS) protocol for the quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin, commonly used antibiotics in intensive care units. The method was previously comprehensively validated according to the guideline of the European Medicines Agency.
After a rapid sample cleanup, the analytes are separated on a C8 reverse-phase HPLC column within 4 minutes and quantified with the corresponding stable isotope-labeled internal standards in electrospray ionization (ESI+) mass spectrometry in multiple reaction time monitoring (MRM). The presented method uses a simple instrumentation setting with uniform chromatographic conditions, allowing for the daily and robust antibiotic therapeutic drug monitoring in clinical laboratories. The calibration curve spans the pharmacokinetic concentration range, thereby including antibiotic amounts close to the minimal inhibitory concentration (MIC) of susceptible bacteria and peak concentrations (Cmax) that are obtained with bolus administration regimens. Without the necessity of the serum dilution before the sample cleanup, the area under the curve for an administered antibiotic can be obtained through multiple measurements.

Using the described protocol, a typical chromatogram is depicted in Figure 2. According to the United States Pharmacopeia (USP) chromatography guidelines16, the column dead volume in the present system was determined with ~0.22 mL and the extra-column volume (including the injector, tubing, and connectors) with ~0.08 mL, giving a hold-up volume of ~0.30 mL. The calculated retention factors for all analytes were 2.8 (for cefepime) - 4.2...

Watch this Scientific Journal Video about Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses at JoVE.com

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

Here we present a tandem mass spectrometry-based protocol for the quantification of frequently used antibiotics in intensive care units, namely cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin.

There is an ever-increasing demand for the therapeutic drug monitoring of antibiotics in many clinical facilities, particularly with regard to the ...

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.

Research

JoVE Journal

Medicine

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Therapeutic Effectiveness of a Dietary Supplement for Management of Halitosis in Dogs

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...