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. For reasons of traceability, insert the manufacturer, catalog number, and a lot number of each antibiotic in the corresponding columns. Dissolve all antibiotics in a cold storage at 4 &#176;C and keep the working time as short as possible.
<ol>
	<li>Prepare 100 mL of 25% methanol in water: prefill a 100 mL volumetric flask with 25 mL of absolute methanol and fill it up to 100 mL with distilled water.</li>
	<li>Prepare 10 mL of 200 mM acetic acid in water: prefill a 10 mL volumetric flask with 9 mL of HPLC grade water, add 115 &#181;L of glacial acetic acid (99.5% purity, 17.4 M), and add distilled water up to 10 mL.</li>
	<li>Prepare 25 mL of 25% methanol in water with 20 mM acetic acid: prefill a 25 mL volumetric flask with 2.5 mL of the aqueous 200 mM acetic acid solution, add 6.25 mL of absolute methanol, and fill up the flask to 25 mL with distilled water.</li>
	<li>Use a precision scale to weigh the proper amounts of antibiotics in 15-mL conical tubes as described in the Supplemental File in the column initial weight.</li>
	<li>Prepare stock solutions of the fluoroquinolones, ciprofloxacin, and moxifloxacin in the 25% methanol-water including 20 mM acetic acid. To do this, add the corresponding volume to the weighted quantities as described in the Supplemental File in the column &#34;final volume&#34;. Rapidly dissolve the fluoroquinolone antibiotics in an ultrasound bath for 2 min and by intense vortexing.</li>
	<li>Prepare stock solutions of cefepime, meropenem, linezolid, and piperacillin in the 25% methanol-water. To do this, add the corresponding volume to the weighted quantities as described in the Supplemental File in the column final volume and rapidly dissolve the antibiotics by intense vortexing. Dissolve meropenem as the last substance.</li>
	<li>Combine the stock solutions of all the antibiotics as described in the corresponding volume of stock solution chart in the Supplemental File to yield tenfold concentrated spike-solutions.</li>
	<li>Spike nine volumes of drug-free serum with one volume of the tenfold concentrated spike solutions to obtain the serum calibrators 0&#8211;7 and quality controls (QC) A&#8211;D. For example, add 0.5 mL of spike solution to 4.5 mL of serum in a 10-mL polypropylene tube and incubate it for 15 min in the cold storage at 4 &#176;C on a roller mixer at 50 rpm.</li>
	<li>Use a repetitive pipette to generate 100 &#181;L aliquots of the calibrators and QCs in 1.5 mL polypropylene tubes.</li>
	<li>Store the calibrators, quality controls, and antibiotic stock solutions at -80 &#176;C for up to six months.</li>
	<li>For each antibiotic, also prepare a neat solution containing 1,000 mg/L of a single antibiotic. Dilute the corresponding stock solution with an appropriate diluent (e.g., for ciprofloxacin, use 25% methanol-water including 20 mM acetic acid). 
	NOTE: The neat antibiotic solutions are required for the instrument-tuning only.</li>
</ol>
2. Preparation of the Internal Standards Mix
NOTE: Internal standards are isotope-labeled counterparts of the analytes of interest that are added to a sample during sample cleanup. As the internal standards have almost identical overall physicochemical properties to their unlabeled counterparts, they compensate for the matrix effects of a given sample.
<ol>
	<li>Prepare 10 mL of 50% methanol in water by adding 5 mL of absolute methanol to a 10 mL shake flask and fill it up to 10 mL with distilled water.</li>
	<li>Prepare 10 mL of 50% methanol in water including 20 mM acetic acid. To do this, add 1 mL of 200 mM acetic acid to a 10 mL flask, add 5 mL of absolute methanol, and fill it up to 10 mL with distilled water.</li>
	<li>Generate stock solutions of internal standards (IS) with 1,000 mg/L directly in the vials provided by the manufacturer. Dissolve cefepime-13C12D3 sulfate in distilled water, meropenem-D6, linezolid-D3, and piperacillin-D5 in a 50% methanol-water solution. Dissolve ciprofloxacin-D8 in 50% methanol-water with 20 mM acetate and moxifloxacin hydrochloride-13C1D3 in distilled water with 20 mM acetate.</li>
	<li>Combine the IS stock solutions in a 1.5 mL polypropylene tube to yield a fivefold concentrated internal standard mix. Add 10 &#181;L of cefepime-13C12D3, 10 &#181;L of meropenem-D6, 1 &#181;L of ciprofloxacin-D8, 2 &#181;L of moxifloxacin hydrochloride-13C1D3, 2 &#181;L of linezolid-D3, and 10 &#181;L of piperacillin-D5 to 965 &#181;L of 25% methanol-water.</li>
	<li>Store the internal standard stock solutions and the fivefold concentrated IS mix at -80 &#176;C.</li>
</ol>
3. Patient Sample Storage
NOTE: Ensure that the serum is obtained as fast as possible and that the cold chain of frozen samples is maintained.
<ol>
	<li>Collect the whole blood in the serum collection tubes.</li>
	<li>Let the blood clot for 20&#8211;30 min at room temperature.</li>
	<li>Separate the serum from the blood by centrifugation at 2,000 x g for 10 min.</li>
	<li>Transfer the supernatant to a clean polypropylene tube.</li>
	<li>Store the serum up to six months at -80 &#176;C until it is assayed. Alternatively, store the samples up to 3 days at -20 &#176;C.</li>
</ol>
4. Buffer Preparation for Chromatography
<ol>
	<li>To prepare 1 M ammonium formate in water, dissolve 6.306 g of ammonium formate in 100 mL of HPLC grade water using a 100 mL shake flask. Store the solution up to 1 month at 4 &#176;C.</li>
	<li>Prepare the mobile phase A [10 mM ammonium formate in water-formic acid (99.9:0.1 v/v)]. Prefill a 1,000 mL volumetric flask with approximately 500 mL of HPLC grade water, add 1 mL of formic acid and 10 mL of the 1 M ammonium formate solution, and fill it to 1,000 mL with HPLC grade water. Transfer mobile phase A to a clean glass bottle and connect it to the HPLC system. Store mobile phase A up to 2 weeks at room temperature.</li>
	<li>Prepare the mobile phase B. Transfer HPLC-grade absolute methanol into a clean glass bottle and connect it to the HPLC system.</li>
	<li>Use absolute methanol as the needle wash solvent and connect the corresponding tube to the glass bottle containing mobile phase B.</li>
	<li>Generate the seal and a purge solvent of methanol-water-formic acid (7:92.9:0.1, v/v/v). Prefill a 1,000 mL volumetric flask with approximately 500 mL of distilled water, add 70 mL of absolute methanol, 1 mL of formic acid, and add distilled water to 1,000 mL. Transfer the solvent to a clean glass bottle and connect it with the HPLC system. 
	Note: Various autosampler systems use both a strong and a weak needle wash solvent. In such a case, prepare the wash solutions according to the manufacturer&#8217;s recommendations. For example, do the strong wash with methanol-water-isopropylic alcohol (70:20:10, v/v/v) and the weak wash with water-methanol (95:5, v/v).</li>
</ol>
5. Instrument Tuning
NOTE: This step is performed for the set-up of the method on a specific mass spectrometer.
<ol>
	<li>Dilute the neat 1,000 mg/L analyte and the internal standard solutions 1:10 or 1:100 in a mixture of mobile phase A and B (50:50, v/v), depending on the detector signal intensities. Tune the mass spectrometer with the autotune function or do a manual tuning for the following parent-to-daughter ions transitions14: cefepime (481.0 &#62; 167.0/395.7), cefepime-13C12D3 (485.1 &#62; 167.1/400.0), meropenem (384.1 &#62; 114.0/141.0), meropenem-D6 (390.1 &#62; 114.0/147.2), ciprofloxacin (332.0 &#62; 231.0/245.0), ciprofloxacin-D8 (340.1 &#62; 235.1/249.3), moxifloxacin (402.0 &#62; 261.0/383.9), moxifloxacin-13C1D3 (406.1 &#62; 265.1/388.0), linezolid (338.0 &#62; 235.0/296.0), linezolid-D3 (341.1 &#62; 235.1/297.1), piperacillin (518.0 &#62; 143.0/358.9), and piperacillin-D5 (523.1 &#62; 142.8/364.1).</li>
	<li>For instruments with autotuning, use the autotune function to automatically adjust the voltage and settings of the MS inlet through the detectors.</li>
	<li>For instruments with manual tuning, adjust the settings (e.g., collision voltage and collision energy) until the optimum (usually the maximum) signal intensity is obtained at the detector for each parent and daughter ion. For example, plug a mixing tee, deliver mobile phase A and B (50:50, v/v) at 0.5 mL/min, and continuously infuse the neat antibiotic or internal standard with a flow rate of 0.1 mL/min.</li>
</ol>
6. HPLC-MS/MS Set-up
NOTE: Features of the mass spectrometer, HPLC system (including the autosampler), and the corresponding software depend on the manufacturer. Adapt the mass spectrometer parameters and the wash procedure according to the manufacturer&#8217;s recommendations.
<ol>
	<li>Store the mass spectrometer parameters in a corresponding &#39;MS tune file&#39;. Use electrospray ionization in the positive mode (ESI+) for all analytes. Adapt the ion source settings for the instrument used (e.g., a capillary voltage of 1.5 kV, a source temperature of 120 &#176;C, a desolvation temperature of 400 &#176;C, a desolvation gas flow rate of 600 L/h, an RF lens voltage of 0.1 V, and a dwell time of 80 ms).</li>
	<li>Specify the analyte and internal standards tune parameters (e.g., capillary voltage, collision energy) in an &#39;MS file&#39;.</li>
	<li>Set the autosampler conditions as follows in the &#39;inlet file&#39;: the sample temperature at 10 &#176;C with a limit of &#177; 5 &#176;C; the wash sequence at 1x purge-wash-purge with a 600 &#181;L purge volume replacement.</li>
	<li>In the above-mentioned &#39;inlet file&#39;, set the flow rate to 0.4 or 0.5 mL/min, the run time to 4 min, the pressure high limit to 345 bar, and the column temperature to 30 &#176;C with a limit of &#177; 5 &#176;C. Add the solvent name of mobile phases A and B and set them to 7% B/93% A, respectively.</li>
	<li>Program the chromatographic gradient in the &#39;inlet file&#39; as follows: 0.00&#8211;0.10 min with 7% mobile phase B/93% A, 0.11&#8211;0.60 min with 65% mobile phase B/35% A, 0.61&#8211;2.10 min with 95% mobile phase B/5% A, 2.11&#8211;4.00 min with 7% mobile phase B/93% A. 
	NOTE: Calculate the extra-column volume, the hold-up volume for the instrumental platform, and the analyte retention factors as described in the USP &#60;621&#62; Chromatography guideline16.</li>
</ol>
7. Sample Measurement Master File
NOTE: With the &#39;sample measurement master file&#39;, the patient samples are specified, the HPLC-MS/MS analysis is started, and the data evaluation is performed. Two separate template files including a low- and high-quality control pair are generated; one template includes QC pair A and C, the other one QC pair B and D.
<ol>
	<li>Create a new &#39;sample measurement master file&#39;. Select the above-mentioned &#39;MS tune file&#39;, &#39;MS file&#39;, and&#160;&#39;inlet file&#39;&#160;(section 6), insert them in each sample line, and specify the injection volume with 15 &#181;L.</li>
	<li>In ascending order, add the &#8220;sample text&#8221; for calibrators 0&#8211;7 and quality control (QC) pair A/C or QC pair B/D.</li>
	<li>Specify the sample type. Select the sample type &#8220;standard&#8221; for the calibrators and &#8220;QC&#8221; for the quality control pairs.</li>
	<li>Specify the concentration of each antibiotic substance for the corresponding calibrators and quality controls (see the spreadsheet, concentration [&#181;g/mL] Cal 7&#8211;Cal 0, QC A/C or B/D,).</li>
	<li>Program the &#39;data evaluation method&#39;.&#160;Use the transitions that were optimized during the instrument tuning (section 5). Match each antibiotic with the corresponding isotope-labeled standard (e.g., meropenem - meropenem-D6).</li>
</ol>
8. Sample Cleanup and HPLC-MS/MS Analysis
NOTE: For each sample batch, a paired quality control set with a low and high antibiotic concentration (QC A/C or QC B/D) is processed and analyzed. Between different batches, the paired QC samples are used in an alternate sequence (e.g., on day 1, select the &#39;sample measurement master file&#39; including QC pair A/C; on day 2, select the one including QC pair B/D. The processing of the serum samples is illustrated in Figure 1.
<ol>
	<li>Prepare the precipitation agent 10% methyl-tert-butyl ether in methanol (10:90, v/v) (e.g., prefill a 25-mL volumetric flask with 2.5 mL of methyl-tert-butyl ether and fill it to 25 mL with absolute methanol).</li>
	<li>Place the C8 reverse phase into the column chamber. Connect it to the HPLC and mass spectrometer in the direction of the flow.</li>
	<li>Generate the sample list. Open the corresponding &#39;sample measurement master file&#39; template and add the patient samples meant to be processed to the list. Generate groups of up to 20 patient samples and flank them with the corresponding quality control pair.</li>
	<li>Wet-prime the HPCL system using the &#39;inlet file&#39; control software: set the &#8220;wet prime&#8221; function to 50% mobile phase A/50% B, and wet-prime for 2 min with a flow rate of 1 mL/min.</li>
	<li>Refresh the syringe. To do this, execute 6 strokes of 600 &#181;L in the control software.</li>
	<li>Equilibrate the C8 reverse phase column. Using the software, turn on the flow in the &#39;inlet file&#39; and flush it with 7% mobile phase B/93% A for a minimum of 5 min, using a flow rate of 0.5 mL/min. Verify the column temperature of 30 &#176;C.</li>
	<li>Thaw the patient samples, one aliquot of calibrators 0&#8211;7, and a quality control pair (either A/B or C/D).</li>
	<li>With a repetitive pipette, add 25 &#181;L of the internal standard mix to the 100 &#181;L calibrator, QC sample, or patient serum in a 1.5 mL polypropylene tube, and vortex the tube for a few seconds.</li>
	<li>Incubate the mixture for 5 min at room temperature on a benchtop shaker (e.g., at 1,200 rpm).</li>
	<li>With a repetitive pipette, add 150 &#181;L of a precipitation reagent to the sample-internal standard mix.</li>
	<li>Again, vortex the tube for a few seconds and incubate it for 5 min at room temperature on a benchtop shaker (e.g., at 1,200 rpm).</li>
	<li>Centrifuge the suspension at 20,000 x g in a tabletop centrifuge for 10 min at 4 &#176;C.</li>
	<li>Dilute the supernatant 1:3 with HPLC grade water using a glass vial with a micro-insert and load it as processed samples to the autosampler.</li>
	<li>Manually start the HPLC-MS/MS analysis in the &#39;sample measurement control file&#39;. 
	NOTE: For prolonged storage, thoroughly flush the analytical column according to the manufacturer&#8217;s recommendation [e.g., 0.5 mL/min methanol-water (50:50, v/v)] to prevent phase collapse.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58148/58148fig1.jpg" /> 
Figure 1: Schematic representation of the sample cleanup. Protein precipitation at the high centrifugal force gives a dense pellet and clear supernatant, indicating that protein precipitation was complete. The entire processing time is approximately 30 min, including the sample cleanup, the chromatographic separation, and the MS/MS analysis. <a href="https://www.jove.com/files/ftp_upload/58148/58148fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
9. Quality Assessment and Quantification
<ol>
	<li>To process the samples, open the corresponding &#39;sample measurement control file&#39;, select the calibrators, quality controls, and patient samples, and evaluate them with the &#39;antibiotics quantification method&#39;. </li>
	<li>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.</li>
	<li>Study the calibration curve and examine whether it fulfills the following quality criteria: a) linearity over the entire calibration range, b) a calibration coefficient r2 &#62; 0.995, c) the deviation of each calibration standard within &#177; 15% of the nominal value, except for the lower limit of quantification (LLOQ), where &#177; 20% is required.</li>
	<li>Reject a calibration standard not complying with the above-mentioned criteria and re-evaluate the calibration curve, including the regression analysis.</li>
	<li>Study the quality controls and examine whether the deviations are within &#177; 15% of the nominal value.</li>
	<li>If the concentration of a patient sample exceeds the concentration of the highest calibrator, dilute the sample with distilled water, up to 1:5 (e.g., 100 &#181;L of serum plus 400 &#181;L of distilled water) before the sample cleanup. Reperform steps 8.8&#8211;8.14 for that specific sample and reprocess it.</li>
</ol>

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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>

The authors have nothing to disclose.

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 ...

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12.8K Views.  LMU Munich. 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.

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

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 ...

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

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. ...

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

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