Trinity College Dublin Doxycycline Hyclate Lab Report

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Title: LC/MS of Doxycycline Analysis 5 Doxycycline belongs to a family of Tetracycline antibiotics that is used in the treatment of a number of infections including lung and respiratory infections. It is also used to treat Lyme disease, acne and some sexually transmitted diseases and can be used in combination with Quinine for the treatment of Malaria. Doxycycline hyclate (DOXH) (C22H24N2O8 xHCl x0.5 C2H5OH x0.5 H2O; molecular mass 512.94 g/mol) is the hydrochloride hemiethanolate hemihydrate salt form which was protonated by HCl and crystallised with 0.5 ethanol and 0.5 water. Anhydrous Doxycycline (DOX) is C22H24N2O8; molecular mass 444.43). DOXH is much more soluble in water than DOX, which is one of the primary reasons for its more frequent use in pharmaceutical preparations. The degradation of doxycycline leads to 6-epi-doxycycline and methacycline as shown below. Mass spectrometry (MS) is an analytical technique used to measure the mass to charge ratio (m/z) of ions to identify and quantify molecules in simple and complex mixtures. Mass spectrometry can be used to determine the molecular structure, molar mass or sample purity and due to its high sensitivity and specificity it is used in many fields of research ranging from drug development to protein analysis. When used in conjunction with liquid chromatography (LC), it provides a highly sensitive tool for quantitative and qualitative analysis. The three main components of a mass spectrometer 1. Ion source: sample molecules are ionized by high voltage 2. Mass analyzer: ions are sorted and separated according to their mass and charge 3. The detector: separated ions are then measured, and the results displayed as a mass spectrum In this practical an electrospray ionization (ESI) is used as the ionizer. Sample flow from the LC enters the ESI nozzle which is held at a positive or negative potential of around 3-5 kV relative to the sample cone depending on the ion polarity. Droplets are formed and the high voltage causes ions of the same polarity come to the solution surface. These ions are released and flow into the mass analyzer. The ions flow through a series of ion optics in the mass analyzer that transmit and focus the ions of interest into the ion trap as a single beam. Any neutral ions or ions of opposite polarity are lost to vacuum, only ions of interest are accumulated in the trap and are then released into the detector. For tandem mass spectrometry (MS/MS), voltages can be applied to cause the ions to fragment into subsequent product ions which give additional qualitative data, the mass spectral fingerprint of the ion. To release the ions from the ion trap, a radio frequency voltage is applied at a low level and then is ramped up so ions are therefore scanned out from low mass to high mass, such that abundance can be measured on an individual ion to ion basis. Electron multiplier detectors collects the ions and converts them to electrons and a current is formed proportional to the amount of ions formed. ----------------------------------------------------------------------------------------------------------- Column Liquid Chromatography Instrument: Thermo Scientific Accela UPLC system Column: Waters Aquity HSS T3-C18 (150mm X 2.1mm, 1.8u) Mobile phase: (A) Water with 0.04% Trifluroacetic acid (B) Methanol:Acetonitrile (50:50) with 0.04% Trifluroacetic acid Isocratic table Time (mins) 0-8 %A 35 %B 65 µl/min 300 Flow rate: 300 µl/min Mass Spectrometry Instrument: LTQ / Orbitrap Discovery Mass Spectrometer (Thermo Scientific) Operating conditions: Electrospray Ionization operated in positive mode and MS/MS of precursor ion at 445 [M+H] with detection of fragmented ion at m/z 428 indicating loss of ammonia [NH3] group [M+H-NH3]. Two scan events were programmed with one full MS scan operated in positive mode and one MS/MS full scan ion trap scan operated in positive mode. Compound MW Ionization method Product ion Doxycycline Hyclate 445 [M+H] positive 428 Positive ionization settings Sheath gas: 70 au Auxiliary gas: 5 Sweep gas: 0 Capillary temp.: 300C Spray Voltage: 3.5 Capillary voltage: 1 Tube Lens: 75 Procedure: Read fully before starting analysis -----------------------------------------------------------------------------------------------------------1. Infusion of DOXH standard directly into the ionisation chamber 1.1 Take 1.0 ml of the 100 µg/ml DOXH stock solution, transfer into a 100 ml volumetric flask and dilute to volume using 0.01 M HCl. 1.2 Using a Hamilton syringe, slowly fill the syringe with 200 µl of sample and connect to PEEK tubing protruding from ESI source of the MS. 1.3 Place syringe into holder and close pusher. Press pump button for two seconds until syringe arm is moving and then release. 1.4 Place MS off standby mode and take account of the observed ion formation on screen. 1.5 Carry out a MS/MS fragmentation of this ion and observe fragments on screen. 2. Injection of DOXH standards into LC-MS Using the 100 µg/ml DOXH stock solution, prepare a set of standards to obtain a calibration curve with linear response to DOXH from 0.5 to 20 μg/mL. 2.1 Prepare six calibration standards with concentrations of 0.5, 1, 5, 7.5, 15, and 20 μg/mL in 10 mL volumetric flasks each using 0.01 M HCl as diluent. Tip: Calculate the volume of stock solution needed for each standard before coming to the practical. 2.2 Filter the standard solutions into 1 mL injection vials using a 0.45 μm filter. 2.3 Load the vials into the autosampler and run sequence (consult a demonstrator) 2.4 When all standards have been injected, obtain their peak areas and plot against concentration using excel, including trendline, equation and r2. 2.5 Record peak area of standards in your Report Sheet and convert DOXH concentration to DOX concentration. Prepare the capsule sample while standards are running. 3. Preparation of DOXH capsules for analysis Capsules were purchased from a local Pharmacy, take note of the label claim. 3.0 Take one DOXH capsule and crush to fine powder using mortar and pestle. 3.1 Add 10 mL of 0.01 M HCl into mortar and mix powder and transfer quantitatively into 100 mL volumetric flask. Rinse any residue in the mortar with 0.01 M HCl and transfer into the volumetric flask. 3.2 Bring to volume with 0.01M HCl and sonicate for five minutes. 3.3 Take 1 mL of this solution and place into 50 ml volumetric flask and dilute to volume with 0.01 M HCl. 3.4 Fill a 2 mL syringe with the diluted sample solution and fit a 0. 45 μm syringe filter. 3.5 Discard the first 2 mL, repeat filtration and collect into 1 mL LC vial, cap it 3.6 Place vial into autosampler and run sequence along with standards. (consult a demonstrator) 3.7 Record the Peak Area of your sample in your Report Sheet and determine the quantity of DOXH/DOX in the tablet using the standard curve least squares method and taking into account the dilutions made. ------------------------------------------------------------------------------------------------------------ Final Report: Calculate the amount of doxycycline in one tablet (remember dilutions!) and compare to the label claim. Sample data will be given to you as part of the online lab in 2021. -----------------------------------------------------------------------------------------------------------Questions: 1. In the chromatogram below, which is taken in the single-ion mode (SIM), two signals can be seen. One is doxycycline and the other is a degradation product. Which of the two degradation products is responsible for this signal? Explain. 2. Hepatic metabolism leads to the formation of 4-epidoxycycline which is inactive. Draw the structure of this epimer. 3. The fragment signal at 428 is due to a loss of ammonia. From the structure of doxycycline, which nitrogen is more likely to be lost during this fragmentation? 05 LC-MS of Doxycycline Standards Concentration DOXH Concentration DOX (512.94 g/mol) (m.w. 444.43 g/mol) Peak Area 0.5 μg/mL 1 μg/mL 5 μg/mL 7.5 μg/mL 15 μg/mL 20 μg/mL Sample Tablet Tablet contains which form of doxycycline (anhydrous / monohydrate / hyclate)? (please circle) Label Claim (anhydrous doxycycline): Sample peak area: Please note any deviations to the procedure PHU22101 Lab Report Experiment 5 – LC-MS of Doxycycline Hyclate Student Name: Date: 1. Introduction to the theory underlying Liquid Chromatography-Mass Spectrometry (brief background to the principles of the technique & instrumentation) → do not write more than one page in this section During the LC-MS/MS experiment (video), the following observations were made: Give an explanation on why the base peak in the spectrum is found at 445. After isolation of the m/z 445 signal in the ion trap, a voltage was applied to induce collision activated dissociation (CAD) = fragmentation. The resulting spectrum shows a base peak at m/z 428. The fragment signal at 428 is due to a loss of ammonia. From the structure of doxycycline, which nitrogen is more likely to be lost during this fragmentation? In the chromatogram below, which is taken in the single-ion mode (SIM), two signals can be seen. One is doxycycline and the other is a degradation product. Which of the two degradation products is responsible for this signal? Explain. Hepatic metabolism leads to the formation of 4-epidoxycycline which is inactive. Draw the structure of this epimer. ____________________________________________________________________________ How were the standard solutions prepared from the stock solution (100 μg/ml DOXH)? Describe with calculation. In the table below, peak areas obtained in this experiment with the standards were inserted in red. Convert the concentration of Doxycyline Hyclate which has a molecular weight of 512.94 g/mol to the concentration of the free base Doxycycline (mw 444.43 g/mol). Show your calculation for one example. Concentration of DOXH (µg/ml) Concentration of DOX (µg/ml) Peak area 0.5 174682 1 398021 5 2739018 7.5 3728643 15 9171940 20 12525608 Plot a graph of the concentration of the DOXH standards against the peak area, including trendline, equation and r2. For the Doxycycline capsule (label claim is 50 mg anhydrous DOX) that was prepared for injection into the LC-MS, calculate the concentration of DOXH (not DOX!) in the solution injected into the LCMS using the equation of the standard curve. Peak area of diluted sample from capsule: 6974171 Calculate the final (theoretical) concentration of Doxycycline DOX (= free base, not the hyclate!) of the injected solution. Using the calculated concentration of DOX in the LC-MS solution, calculate the amount of Doxycycline in one capsule and compare your result to the label claim (50 mg anhydrous DOX). What do you think you have learnt in this lab/when writing up this lab report? • • • • • Plagiarism statement:
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PHU22101 Lab Report
Experiment 5 – LC-MS of Doxycycline Hyclate
Student Name:
Date:

1. Introduction to the theory underlying Liquid Chromatography-Mass Spectrometry
The first precise measurement of ionic masses was carried out at the beginning of the 20th century
by Aston and Dempster. Today, mass spectrometry represents one of the most often used
instrumental techniques in precise measurements of thousands of compounds in many areas of
science. The basic principle of mass spectrometry (MS) is the ability to measure the mass to charge
(m/z) ratio of the compound. The first mass spectrometry instruments were combined with gas
chromatography. The main challenge was to combine the liquid chromatography (LC) and MS.
Namely, as the compounds of interest were dissolved in liquid during the chromatographic
separation by liquid chromatography, the development of ionization techniques (e.g. electrospray
ionization) helped in coupling the LC to MS. The most important parts of LC-MS instruments are
sample introduction equipment and chromatography column, ion source, a mass analyzer, and
detector [1]. Besides the determination of the m/z ration, MS is capable to determine the molecular
structure as well as sample purity [2].
The main purpose of LC devices is to enable efficient separation of matrix components in time.
Different compounds present in the sample matrix leave the column of LC devices and comes to the
ion source. The gradient program of LC devices helps in the chromatographic separation of
compounds present in the sample matrix [1,2].
Different ionization techniques have been developed in the past in order to enable the ionization of
compounds of interest. The most often used ionization techniques in LC-MS are ESI, atmospheric
pressure photo-ionization (APPI), atmospheric pressure chemical ionization (APCI), and Matrixassisted laser desorption/ionization (MALDI) [1].
Electrospray is produced when a strong electric field (2.5 to 5 kV at low potential 0 to 100 V) is
applied at the end of the (heated) capillary tube. In this way, the fine droplets are formed. During a
short period of time, the volume of drops decreases as a consequence of the evaporation of solvent
from the surface of the droplets. The evaporation of the solvent is supported by desolvation gas (e.g.
...


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