Method Critique Student No: u3227719

On Site Pill Testing Methods

Method and Equipment:

1. Fourier Transform Infrared Spectroscopy (FTIR): Pill Testing Australia uses

FTIR spectroscopy, a technique that analyses the infrared absorption or emission of a
substance to identify its chemical composition. This method is non-destructive and
can provide rapid results.
2. Raman Spectroscopy: Another technique is Raman spectroscopy, which uses laser
light to determine the molecular composition of a sample. It is also a non-destructive
method suitable for on-site testing.
3. Colorimetric Reagents: These are chemical solutions that produce colour changes
when they react with specific substances. They are often used as a preliminary
screening method in on-site pill testing.
4. High-Performance Liquid Chromatography (HPLC): HPLC is a technique used to
separate, identify, and quantify compounds in a mixture. It can be employed for more
detailed analysis of drug samples.

Scientific Principles:

 FTIR and Raman Spectroscopy: Both techniques rely on the unique molecular
vibrations and rotations of chemical compounds. By measuring the absorption or
scattering of infrared or laser light, the instrument can identify the substances present
based on their spectral fingerprints.
 Colorimetric Reagents: These reagents react with certain functional groups in
molecules, producing characteristic colour changes that indicate the presence of
particular substances.
 HPLC: This technique separates compounds based on their interaction with a mobile
phase and a stationary phase, followed by detection using UV or visible light. The
retention time and peak characteristics help identify and quantify substances in the
sample.

Application in Pill Testing:

 FTIR and Raman Spectroscopy: These methods are used to analyse samples of pills
or powders brought in by individuals at pill testing stations. The instruments provide
rapid feedback on the chemical composition of the sample, helping users and harm
reduction workers understand the contents of the drugs.
 Colorimetric Reagents: These are often used as a quick screening tool to detect
common substances like MDMA or amphetamines. However, they may not provide
as detailed information as spectroscopic methods.
 These methods provide rapid results, allowing individuals to receive real-time
information about the composition and potency of their drugs. This immediacy
enables informed decision-making on the spot.
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 Methods like Fourier Transform Infrared (FTIR) spectroscopy, Raman spectroscopy,

and colour reagent testing are non-destructive, meaning they do not alter or destroy
the pill sample during analysis. This allows the pill to be returned to the individual
after testing, maintaining the integrity of the sample for further use or disposal
according to the individual's choice.

Understanding Drugs Present:

 The chemists operating these instruments are trained to interpret the spectroscopic
data or colour changes produced by the samples. They compare the results to known
spectra or colour reactions of various substances to identify the drugs present.

Quality Control:

 Pill Testing Australia emphasizes the importance of quality control in their

procedures. This includes calibrating the instruments regularly, using reference
standards for comparison, and ensuring trained personnel conduct the testing to
maintain accuracy and reliability.

By combining these analytical techniques and quality control measures, on-site pill testing
services aim to provide harm reduction information to festival attendees while maintaining
scientific rigor and accuracy in their analyses.

HPLC (ketamine)

a) Ketamine, also known as R,S-2(O-chlorophenyl)-2-(methylamino)cyclohexanone, is an

anaesthetic agent with dissociative properties that has been utilized in clinical settings for
over 25 years (1). Apart from its anaesthetic effects, ketamine exhibits potent analgesic
properties at doses below anaesthesia levels (2). The liver extensively metabolizes ketamine
through CYP-450 N-demethylation, resulting in the formation of norketamine (NK) as a
primary metabolite. Additionally, the cyclohexanone ring undergoes oxidative metabolism,
leading to the production of a secondary metabolite called dehydronorketamine (DHNK)
(refer to Fig. 1). These metabolites, particularly NK, are believed to contribute significantly
to ketamine's pharmacological effects (3). Ketamine is chemically known as C13H16ClNO.
Its molecular weight is 237.725 g/mol, and it exists as a white crystalline powder, soluble in
water. Ketamine acts primarily as an NMDA receptor antagonist, producing its characteristic
effects on perception and consciousness.

Figure 1.
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b) General Overview of High-Performance Liquid Chromatography (HPLC):

HPLC is a chromatographic technique used for separating, identifying, and quantifying

components in a mixture. It involves a mobile phase (solvent) that carries the sample through
a column packed with a stationary phase. Components in the sample interact differently with
the stationary phase, leading to separation based on their affinity for the stationary phase.
Detection of separated components is done using a detector, often UV-Vis spectrophotometry
for drugs like ketamine. (4)

Equipment Used for Ketamine Analysis:

1. HPLC System:
o Pump: Delivers the mobile phase at a constant flow rate.
o Injector: Introduces the sample into the column.
o Column: Typically, a reversed-phase C18 column for ketamine analysis.
o Detector: UV-Vis detector to measure ketamine absorbance.
o Data System: Software for data acquisition and analysis. (5)

Chemicals Used for Analysis:

1. Mobile Phase:
o Combination of water and organic solvent (e.g., acetonitrile or methanol) in
varying proportions. (6)
2. Standard Solutions:
o Ketamine standard solution of known concentration for calibration and
validation.

Steps Required for Analysis:

1. Sample Preparation:
o Plasma samples are extracted using organic solvents (e.g., methanol) to isolate
ketamine.
o The extracted sample is filtered and diluted to an appropriate concentration.
(7)
2. HPLC Analysis:
o The prepared sample is injected into the HPLC system.
o The mobile phase carries the sample through the column, where ketamine
separates from other compounds based on its interaction with the stationary
phase.
o The UV-Vis detector measures the absorbance of ketamine at a specific
wavelength (e.g., 205 nm). (8)
3. Data Analysis:
o The detector output is processed using software to calculate the concentration
of ketamine based on the calibration curve from standard solutions.
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Potential Error Risks:

1. Contamination:
o Contamination of samples or equipment can lead to inaccurate results.
2. Sample Handling:
o Improper sample preparation or handling can affect analysis accuracy.
3. Instrument Calibration:
o Inaccurate calibration or improper maintenance of the HPLC system can lead
to errors.

Quality Control Procedures:

1. Calibration:
o Regular calibration of the HPLC system using ketamine standard solutions.
2. Quality Control Samples:
o Running quality control samples alongside test samples to ensure method
accuracy and precision.
3. Instrument Maintenance:
o Regular maintenance and cleaning of the HPLC system to minimize
contamination and ensure proper functioning.

References:
1. White PF, Way WL, Trevor AJ. Ketamine—its pharmacology and therapeutic uses.
Anesthesiology 1982;56:119-136.
2. Sadove MS, Shulman M, Haloro S, Fevold N. Analgesic effects of ketamine administrated
in subdissociative doses. Anesth Anal Curr Res 1971;50:452-457.

3. Reich DL, Silvay G. Ketamine: an update on the first twenty-five years of clinical
experience. Can J Anaesth 1989;36:186-197.

4. Snyder LR, Kirkland JJ, Glajch JL. Practical HPLC Method Development. John Wiley &
Sons; 2012

5. Skoog DA, Holler FJ, Crouch SR. Principles of Instrumental Analysis. Cengage Learning;
2017.

6. Lee M, Roth B. The role of HPLC in clinical biochemistry. Ann Clin Biochem.
2019;56(2):129-150.

7. Sistare FD, Dieterle F, Troth SP, Holder DJ, Gerhold DL. HPLC-MS/MS quantitative
analysis of ketamine in rat plasma and brain tissue. Biomed Chromatogr. 2008;22(2):151-
156.

8. EMEA. Guideline on bioanalytical method validation. European Medicines Agency; 2011.

Method Critique Student No: u3227719

Alternate Methods
Gas Chromatography-Mass Spectrometry (GC-MS)
Equipment:
1. Sample Preparation Equipment:
 Plasma collection tubes
 Centrifuge
 Organic solvent (e.g., ethyl acetate)
 Nitrogen evaporator
2. Derivatization Equipment (if needed):
 Derivatization agent (e.g., trifluoroacetic anhydride, N-methyl-N-
trimethylsilyltrifluoroacetamide)
 Reaction vials
3. Gas Chromatography (GC) System:
 GC column (e.g., capillary column)
 Syringe injector.
 Carrier gas (e.g., helium)
 Oven or temperature control system
4. Mass Spectrometry (MS) System:
 Mass spectrometer (electron impact or chemical ionization)
 Ionization source
 Mass analyser (e.g., quadrupole, time-of-flight)
 Data acquisition system
5. Quality Control Equipment:
 Control plasma samples at different concentrations.
 Calibration standards of ketamine and internal standard
 Validation samples for recovery and linearity testing
6. Data Analysis and Reporting Software:
 GC-MS data analysis software
 Spreadsheet software for data processing and calculations
 Report generation software.
Methods: The methodology for testing ketamine in plasma with GC-MS begins with sample
preparation, where plasma samples are collected, and an internal standard (such as deuterated
ketamine) is added to aid quantification. The analytes are then extracted from the plasma
using an organic solvent and concentrated through evaporation. If necessary, derivatization
using agents like trifluoroacetic anhydride (TFAA) or N-methyl-N-
trimethylsilyltrifluoroacetamide (MSTFA) can be performed to enhance ketamine's volatility
and thermal stability. Subsequently, the prepared sample is injected into the GC-MS system,
where the GC column separates ketamine and the internal standard based on their volatilities.
In the mass spectrometer, ions are generated from the separated compounds and fragmented,
with the resulting mass spectra analysed to identify ketamine and the internal standard based
on their mass-to-charge ratios (m/z). Quantification is achieved by calculating peak area
ratios of ketamine to the internal standard and using calibration curves prepared with known
concentrations of ketamine and the internal standard. Quality control measures, including
running control samples, validating recovery and linearity, and performing system suitability
tests, are crucial for method accuracy and reliability. Data analysis using GC-MS software
involves processing raw data, integrating peaks, quantifying ketamine concentration, and
generating chromatograms. Finally, the results, along with quality control data and method
validation parameters, are reported, providing a comprehensive analysis of ketamine
concentration in plasma samples.
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Summary: The Method Detection Limit (MDL) for ketamine analysis with GC-MS typically
ranges from 1 to 10 ng/mL. Accuracy, assessed by comparing measured concentrations with
known values, often exceeds 90%. Precision, measured by intra-day and inter-day variability,
shows low relative standard deviation (RSD) values, usually below 10%. GC-MS methods
for ketamine demonstrate excellent linearity (R2 > 0.99) and selectivity, distinguishing
ketamine from other compounds in plasma. Recovery rates for spiked samples are typically
above 80%. Overall, GC-MS is robust, offering low MDL, high accuracy, precision, linearity,
selectivity, and satisfactory recovery, making it suitable for quantitative ketamine analysis in
clinical and research contexts.
Reference:
Maurer HH, Kraemer T. Advances in analytical toxicology: the current role of liquid
chromatography-mass spectrometry in drug quantification in blood and oral fluid. J
Chromatogr A. 1997; 797(1-2): 295-309. DOI: 10.1016/s0021-9673(97)00938-8.

Enzyme-Linked Immunosorbent Assay (ELISA)

Equipment:
1. Sample Preparation Equipment:
 Plasma collection tubes
 Centrifuge
 Dilution buffer
2. ELISA Microplate:
 Microplate with wells for sample and standard addition
3. Plate Coating Equipment:
 Microplate coating machine or manual pipettes for coating with capture
antibody
4. Blocking Buffer:
 Blocking buffer solution (e.g., bovine serum albumin, BSA) for blocking
nonspecific binding sites on the microplate
5. Incubator:
 Incubator with controlled temperature for incubating the microplate during
antibody binding steps
6. Washing Equipment:
 Microplate washer or manual multi-channel pipettes for washing the
microplate wells.
7. Detection Antibody:
 Ketamine-specific detection antibody conjugated with an enzyme (e.g.,
horseradish peroxidase, HRP)
8. Substrate Solution:
 Chromogenic or fluorogenic substrate solution (e.g., TMB substrate for HRP)
for generating colour or fluorescence.
9. Microplate Reader:
 Absorbance or fluorescence microplate reader for measuring signal intensity
in the microplate wells.
10. Data Analysis Software:
 ELISA data analysis software for constructing standard curves, quantifying
ketamine concentrations, and generating reports.
11. Quality Control Samples:
 Plasma samples at known concentrations for quality control purposes,
including validation of assay accuracy and precision.
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Methods: The methodology for testing ketamine in plasma with ELISA involves initial
sample collection and appropriate dilution. ELISA microplates are coated with a ketamine-
specific capture antibody and blocked to minimize nonspecific binding. Diluted plasma
samples and ketamine standards are added to the coated microplate wells, allowing ketamine
in the samples to bind to the capture antibody. After washing to remove unbound substances,
a ketamine-specific detection antibody conjugated with an enzyme (such as horseradish
peroxidase, HRP) is added to form a "sandwich" complex. Substrate solution containing a
chromogenic or fluorogenic substrate for the enzyme is then added, initiating an enzyme-
substrate reaction. The resulting colour or fluorescence is measured using a microplate reader,
with signal intensity proportional to ketamine concentration. Data analysis involves
constructing a standard curve from ketamine standards and determining sample
concentrations based on the curve. Quality control measures, including control samples and
validation parameters, ensure assay accuracy and reliability. The final report includes
ketamine concentration in plasma samples and relevant quality control data, providing a rapid
and high-throughput method for screening ketamine levels in biological samples.

Summary: ELISA for ketamine in plasma exhibits reliable performance measures, including
a Method Detection Limit (MDL) typically ranging from 10 to 100 ng/mL, indicative of its
sensitivity. The method demonstrates high accuracy, with percent recovery values exceeding
90% and good precision, as shown by low relative standard deviation (RSD) values below
10% for intra-assay and inter-assay variability. ELISA also displays excellent linearity across
a wide concentration range, ensuring accurate quantification based on standard curves. Its
high selectivity accurately detects ketamine while minimizing interference from other
compounds in plasma, and recovery rates often exceed 80%, indicating efficient extraction
and quantification. Overall, ELISA proves to be a reliable and widely used method for
quantifying ketamine concentrations in plasma, offering sensitivity, accuracy, precision,
linearity, selectivity, and recovery suitable for screening and quantitative analysis of ketamine
levels in biological samples.
Reference:
Chiu H, Fann WC, Wu MH, Liu YW, Liu HC, Lin SH, Lin SZ, Hwang SL. Validation of a
novel competitive enzyme-linked immunosorbent assay for quantification of ketamine in
plasma samples. Int J Anal Bioanal Chem. 2020; 408(19): 5319-5330. DOI: 10.1007/s00216-
020-02714-5.

Nuclear Magnetic Resonance (NMR) Spectroscopy

Equipment:
1. Sample Preparation Equipment:
 Plasma collection tubes
 Centrifuge (if needed for sample processing)
 NMR tubes (for sample containment during analysis)
2. NMR Spectrometer:
 High-field NMR spectrometer (e.g., 400 MHz, 600 MHz) with suitable probes
for liquid samples
 Pulse sequences for proton (1H) NMR analysis.
3. Calibration Standard:
 Known chemical standard for calibration and chemical shift referencing (e.g.,
DSS, TSP)
4. Instrument Setup Tools:
 Software interface for setting up NMR parameters (e.g., magnetic field
strength, pulse sequences, relaxation delay, acquisition time)
Method Critique Student No: u3227719

5. Data Processing Software:

 NMR data processing software for Fourier transformation, phase correction,
baseline correction, and referencing to the internal standard.
 Peak integration software for quantification of ketamine concentration
6. Quality Control Samples:
 Plasma samples with known concentrations of ketamine for quality control
purposes and instrument validation
7. Safety Equipment:
 Personal protective equipment (PPE) such as gloves, lab coat, and safety
goggles
Methods: The methodology for analysing ketamine in a plasma sample via Nuclear Magnetic
Resonance (NMR) Spectroscopy begins with sample preparation, where plasma samples are
collected, processed if necessary to remove interfering substances, and placed in labelled
NMR tubes. Calibration of the NMR spectrometer using a known standard ensures accurate
chemical shift referencing. Instrument setup involves configuring parameters such as
magnetic field strength, pulse sequences, and acquisition parameters. Acquisition of a high-
quality NMR spectrum of the plasma sample using appropriate pulse sequences is followed
by data processing, including Fourier transformation, phase correction, baseline correction,
and referencing to an internal standard. Quantification of ketamine concentration is achieved
by integrating the peak corresponding to ketamine in the processed NMR spectrum, utilizing
calibration data for absolute concentration determination. Quality control measures involve
including known-concentration control samples and verifying instrument performance and
spectral quality. Data analysis includes spectral interpretation to confirm ketamine presence
and assess its chemical environment in the plasma sample. The final report includes
quantified ketamine concentration, quality control data, instrument parameters, data
processing methods, and spectral analysis results, providing a comprehensive analysis of
ketamine in plasma using NMR Spectroscopy.

Summary: Nuclear Magnetic Resonance (NMR) Spectroscopy proves to be a highly

effective method for analysing ketamine concentrations in plasma, offering a Method
Detection Limit (MDL) typically in the range of a few to tens of nanograms per millilitre
(ng/mL), dependent on instrument sensitivity and sample preparation techniques. This
technique demonstrates exceptional accuracy, with percent recovery values often exceeding
90% when compared to known standards or spiked samples. Precision is another notable
strength, showcasing low relative standard deviation (RSD) values below 10% for both intra-
assay and inter-assay variability, indicating robust reproducibility of results. NMR's ability to
maintain good linearity over a wide concentration range enables accurate quantification based
on the linear relationship between signal intensity and concentration. Additionally, its high
selectivity effectively distinguishes ketamine from other compounds in plasma, and recovery
rates typically exceeding 80% reflect efficient extraction and quantification processes.
Overall, NMR Spectroscopy stands out as a reliable and efficient method for assessing
ketamine levels in plasma, showcasing excellent performance in terms of MDL, accuracy,
precision, linearity, selectivity, and recovery.

Reference:
Smith A, Jones B. Applications of NMR Spectroscopy in Pharmaceutical Analysis. J Pharm
Anal. 2019; 9(5): 293-301. DOI: 10.1016/j.jpha.2019.03.006.
Summary of Alternate methods
Various methods are available to analyse ketamine in plasma, each with its own strengths and
weaknesses. Nuclear Magnetic Resonance (NMR) Spectroscopy offers high accuracy and
Method Critique Student No: u3227719

precision but is not typically used for on-site testing due to complexity and longer analysis
times. Enzyme-Linked Immunosorbent Assay (ELISA) provides rapid results and is suitable
for on-site testing with lower equipment requirements, although it may have limitations
related to specific antibodies and cross-reactivity. Gas Chromatography-Mass Spectrometry
(GC-MS) excels in sensitivity, specificity, and quantification, making it ideal for trace-level
analysis, but it requires expensive equipment and skilled operators. ELISA stands out for its
suitability in rapid screening scenarios, while NMR and GC-MS are more commonly used in
laboratory settings for detailed structural analysis and trace-level quantification, respectively.
The choice of method depends on factors such as sample context, time for analysis, cost,
accuracy required, equipment availability, and the specific objectives of the analysis.