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>
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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.

multiplex-therapeutic-drug-monitoring-isotope-dilution-hplc-msms

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 manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

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 sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load &#62;1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads &#60;1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

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 limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects 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 ...

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 mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

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

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

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 model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

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 insufficient oxygen supply and irreversible organ failure. The brain exerts only limited compensation capacities and is particularly at high risk of severe hypoxic damage.This article demonstrates the reproducible induction of life-threatening hemorrhagic shock in a porcine model by means of calculated blood withdrawal. We titrate shock induction guided by near-infrared spectroscopy&#160;and extended hemodynamic monitoring to display systemic circulatory failure, as well as cerebral microcirculatory oxygen depletion. In comparison to similar models that primarily focus on predefined removal volumes for shock induction, this approach highlights a titration by means of the resulting failure of macro- and microcirculation.

Hemorrhagic shock is a severe complication in seriously injured patients, which leads to life-threatening oxygen undersupply. We present a standardized method to induce hemorrhagic shock via blood withdrawal in pigs that is guided by hemodynamics and microcirculatory cerebral oxygenation.

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 therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

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 luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

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. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

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

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been implicated in impacting long-term cancer outcomes, possibly through modulation of adrenergic-inflammatory responses that impact cancer cell behavior and immune cell function. Emerging evidence suggests that propofol-based total intravenous anesthesia (TIVA) may be beneficial for long-term cancer outcomes when compared to inhaled volatile anesthesia. However, the available clinical findings are inconsistent. Preclinical studies that identify the underlying mechanisms involved are critically needed to guide the design of clinical studies that will expedite insight. Most preclinical models of anesthesia have been extrapolated from the use of anesthesia in in vivo research and are not optimally designed to study the impact of anesthesia itself as the primary endpoint. This paper describes a method for delivering propofol-TIVA anesthesia in a mouse model of breast cancer resection that replicates key aspects of clinical delivery in cancer patients. The model can be used to study mechanisms of action of anesthesia on cancer outcomes in diverse cancer types and can be extrapolated to other non-cancer areas of preclinical anesthesia research.

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

This paper describes a method for modeling total intravenous anesthesia (TIVA) during cancer resection surgery in mice. The goal is to replicate key features of anesthesia delivery to patients with cancer. The method allows investigation of how anesthetic technique affects cancer recurrence after resection surgery.

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been ...