Spectrometric Detection: UV-Vis, FTIR and Mass Spectrometry

UV-Vis is the oldest molecular spectrometry technique on the toxicology bench and remains the most frequently run. A xenon or deuterium-tungsten source, a monochromator, a quartz cell and a photomultiplier produce an absorbance reading in under thirty seconds per sample. The reading converts to a concentration through the Beer-Lambert law: A = εbc, where A is absorbance, ε is the molar absorptivity in litres per mole per centimetre, b is path length (almost always 1 cm in a standard cuvette) and c is concentration in moles per litre. The relation is linear up to absorbances around 1.0 in most instruments, beyond which detector saturation and stray light push readings off the curve.

The applications fall into a handful of routine patterns. Paracetamol in serum is read at 245 nm after alkaline extraction; the alkaline shift moves the phenolate absorbance to a useful maximum and pulls it away from interferents. Barbiturates show a characteristic 240 nm peak in alkaline solution that shifts on protonation, and the pH-dependent shift itself is diagnostic. Salicylate quantitation rides on the Trinder reaction, where ferric chloride produces a violet iron-salicylate complex read at 540 nm; the reaction is the basis of both bedside spot tests and routine emergency-room quantitation. Morphine in alkaline solution gives a maximum near 285 nm.

The diode array detector (DAD) variant has replaced single-wavelength UV-Vis on most state laboratory benches over the last decade. A DAD captures the full 190 to 800 nm spectrum in under a second, which means an analyst can read a sample once and see every chromophore in the cuvette. The spectrum itself becomes a fingerprint: the ratio of 240 nm to 280 nm absorbances, the wavelength of maximum absorbance, the shape of the peak. Library matching against a DAD reference set is a useful presumptive identification even before any chromatography or mass spectrometry runs.

The catch with UV-Vis is specificity. Most drugs absorb somewhere between 200 and 300 nm. Serum proteins, bilirubin, haemoglobin and a long list of endogenous chromophores absorb in the same window. A 245 nm peak in a serum extract is consistent with paracetamol, but it is also consistent with a dozen other compounds and a few common matrix contaminants. The technique gives an excellent concentration when the analyte identity is known; it gives a poor identification when the analyte identity is in question. That single limitation is why UV-Vis sits below mass spectrometry in every confirmatory hierarchy.

Fourier-transform infrared spectroscopy is the standard technique for identifying an unknown solid or liquid. A Michelson interferometer modulates the infrared beam, the sample absorbs at characteristic vibrational frequencies, and a fast Fourier transform converts the interferogram into a transmittance spectrum across roughly 4000 to 400 cm⁻¹. The whole scan takes about ninety seconds on an ATR instrument and produces a spectrum that is genuinely diagnostic.

The sampling mode used to matter a lot. KBr pellet preparation (1 to 2 mg of analyte ground with 100 to 200 mg of dry KBr, pressed at 10 tons into a transparent disc) was the standard for solids through the 1990s. Nujol mull (a paste of analyte in mineral oil between salt plates) was the workhorse for oily liquids and moisture-sensitive samples. Both required practice and a steady hand. ATR, with its diamond or ZnSe crystal pressed against the sample, has replaced almost all routine pellet and mull work. The evanescent wave probes only the first few micrometres of the sample, so the ATR spectrum is surface-biased, but for forensic identification of a tablet, powder, paint chip or residue that is exactly what you want.

The information lives in two regions of the spectrum. Above 1500 cm⁻¹ sit the group frequencies: a carbonyl C=O near 1700 cm⁻¹, a broad OH between 3200 and 3600 cm⁻¹, an NH between 3300 and 3500 cm⁻¹, aromatic C-H between 3000 and 3100 cm⁻¹, aliphatic C-H between 2850 and 2960 cm⁻¹. These tell you what classes of functional groups are present. Below 1500 cm⁻¹ sits the fingerprint region: a dense pattern of bending and combination bands that is essentially unique to each compound. Library matching is dominated by the fingerprint region. A match score above 0.95 against a reputable database (Mainlib, Sadtler, the SWGDRUG infrared library) is generally accepted as confirmatory identification in Indian and international forensic practice.

Forensic applications span several evidence categories. An unknown white powder in a seized exhibit may be paracetamol, ammonium nitrate, methamphetamine, sugar, or talc. FTIR distinguishes them in a single scan. Tablets recovered from a suicide note can be matched against a reference library to confirm whether they are the prescription on the bedside table or something else. Explosive residues (TATP, RDX, PETN) have signature FTIR spectra catalogued in the National Bomb Data Centre and NSG forensic libraries. Counterfeit pharmaceuticals are routinely identified at the Central Drugs Standard Control Organisation (CDSCO) using FTIR comparison to an authenticated reference. Paint chips from a hit-and-run can be matched to a vehicle through layered FTIR of the binder and pigment.

1. Background scan
   Run a clean background spectrum on the empty ATR crystal. Save it as the reference. Skipping this step pulls atmospheric CO2 and water vapour bands into the analyte spectrum and ruins the match score.
2. Sample presentation
   Press a small portion of the unknown powder, tablet shaving or liquid film against the diamond ATR crystal. The pressure clamp ensures intimate contact; a poor contact gives a weak signal and tilted baseline.
3. Acquire the spectrum
   16 to 32 scans at 4 cm⁻¹ resolution between 4000 and 400 cm⁻¹. Total acquisition time is under two minutes. Save the .spc or .csv file with the case number and sample ID baked into the filename.
4. Baseline correct and library search
   Auto-baseline-correct, then run a library search against Mainlib, Sadtler and any in-house reference set. Sort hits by match score. A match above 0.95 with a chemically plausible top hit is a confirmed identification; a match between 0.85 and 0.95 needs orthogonal confirmation by GC-MS or LC-MS.
5. Report with the matched spectrum
   The report appends the sample spectrum overlaid on the library reference with the match score, the library used, the version of the search algorithm and the analyst's initials. Without the overlay the result is not defensible at trial.

One important limitation concerns mixtures. FTIR is a bulk technique. If the unknown is a mixture of two or more components, the spectrum is a sum of the component spectra weighted by their mass fractions. Library matching against a mixture typically gives a top hit at a low score (0.60 to 0.85) and several plausible runners-up. The correct approach at that point is microscopy combined with separation: isolate individual particles under a stereo microscope, or run HPLC or TLC, then acquire an ATR spectrum of each component. Reporting the mixture spectrum's top hit without component isolation is a recurring source of identification error.

Mass spectrometry is the confirmatory tier: presumptive findings from immunoassay, colour tests, or UV-Vis must be confirmed by MS before being reported as a positive case finding. Every modern toxicology SOP, from SAMHSA in the United States to SOFT international guidelines to the operating protocols of CFSL Chandigarh and FSL Madhuban, treats MS as the confirmatory tier. The technique ionises the analyte, separates ions by mass-to-charge ratio (m/z), and counts them at a detector. The three working instruments differ in how the analyte enters the source, how it is ionised, and how the analyser separates the ions.

GC-MS is the oldest and still the gold standard for volatile and semi-volatile organics. The sample is injected into a gas chromatograph, separated on a capillary column, then enters the MS source. Ionisation is electron impact (EI) at 70 eV, a hard ionisation that fragments the molecule extensively and reproducibly. The fragmentation pattern is matched against the NIST or Wiley library; a match factor above 800 (out of 1000) with a chemically plausible top hit is generally accepted as identification. GC-MS covers the classical toxicology panel: ethanol and methanol, organophosphate pesticides (after extraction), benzodiazepines and tricyclics (after derivatisation with BSTFA or MSTFA), opiates and their metabolites, and amphetamines. Every Tier-1 SFSL in India runs GC-MS as a routine instrument, with Agilent 5977 and Shimadzu QP2020 the two commonest workhorses on the Indian bench at CFSL Chandigarh, CFSL Hyderabad, FSL Madhuban and FSL Kalina Mumbai.

LC-MS/MS is the modern toxicology workhorse for non-volatile and thermally labile drugs. The sample is separated on a reversed-phase liquid chromatography column, then ionised by electrospray ionisation (ESI) or atmospheric pressure chemical ionisation (APCI). ESI is the default, a soft ionisation that produces a protonated molecular ion [M+H]+ with little fragmentation, ideal for fragile drugs and metabolites. The triple-quadrupole analyser (QqQ) then runs in multiple reaction monitoring (MRM) mode: Q1 filters the precursor ion, Q2 fragments it with collision gas (argon or nitrogen), Q3 filters a chosen product ion. Each precursor-to-product pair is one transition. LOQs of 0.1 to 1 ng/mL in plasma or urine are routine, with some assays reaching into the low picogram range. Quantitation is performed against a deuterated internal standard (d3-morphine, d5-EDDP, d3-cocaine, d5-alprazolam) spiked at a known concentration into every sample.

High-resolution mass spectrometry (HRMS), in the form of Q-TOF or Orbitrap instruments, represents the current advanced tier on the Indian forensic bench. Mass accuracy below 5 parts per million allows an unknown ion to be assigned to a unique elemental composition; that composition, combined with isotope-pattern matching and MS/MS fragmentation, is enough to identify novel psychoactive substances (NPS) and unknown plant toxins that have never been added to any library. The instruments are concentrated at the research and reference centres: NIPER Mohali for pharmaceutical analysis, the National Centre for Biological Sciences (NCBS) Bangalore for metabolomics, the National Dope Testing Laboratory (NDTL) Delhi for anti-doping. A routine SFSL toxicology bench rarely needs HRMS; an emerging-drug case in a state under WADA jurisdiction routinely does.

The cross-confirmation rule is brief and non-negotiable. A presumptive immunoassay screen (or a UV-Vis spot, or a TLC band) is never reported as a confirmed identification. Confirmation requires a chromatographic separation followed by mass spectrometry with at least two diagnostic ion transitions (for LC-MS/MS in MRM mode) or a full-scan EI spectrum library match above 800 (for GC-MS), plus a retention-time match within roughly ±2 percent of a calibrator. The MRM transition ratio (the ratio of qualifier to quantifier ion areas) must agree with the calibrator within ±20 percent for low concentrations and ±10 percent at higher concentrations. A deuterated internal standard runs in every sample to control for matrix effects and injection variability. Omitting any of these steps produces a result that may be scientifically accurate but is legally vulnerable to challenge.

Three ionisation modes cover the great majority of toxicology MS applications. The choice is dictated by the physical properties of the analyte.

Electron impact (EI) at 70 eV is the GC-MS standard. A heated filament emits electrons, the molecule passes through the beam in the gas phase, an electron-molecule collision strips an electron and leaves an unstable molecular radical cation. The radical cation fragments along well-defined bond-cleavage pathways, and the resulting fragment pattern is reproducible across instruments and decades. That reproducibility underpins the NIST library. Match an unknown EI spectrum against the library and a top hit with score above 800 plus a sensible top-three is usually a confirmed identification. EI gives extensive fragmentation; the molecular ion is sometimes weak or absent for fragile molecules, which is why some workflows use chemical ionisation (CI) as a complementary mode to confirm molecular weight.

Electrospray ionisation (ESI) is the LC-MS standard. The liquid eluent from the chromatograph is sprayed through a charged capillary, and the resulting droplets evaporate until the analyte ions are ejected into the gas phase. The mode is soft, with little fragmentation; the analyst sees the protonated molecule [M+H]+ as the dominant ion in positive mode, or [M-H]- in negative mode. Drugs with basic nitrogen (morphine, cocaine, amphetamines, benzodiazepines) ionise efficiently in positive ESI. Drugs with acidic groups (THC-COOH, salicylate, barbiturates) ionise in negative mode. ESI is widely used because almost any drug small enough to elute from a reversed-phase column will ionise under one polarity or the other.

Atmospheric pressure chemical ionisation (APCI) sits between EI and ESI. A heated nebuliser vaporises the LC eluent, a corona discharge ionises a reagent gas, and proton transfer from the reagent to the analyte produces [M+H]+. APCI handles semi-volatile, less polar analytes that ionise poorly under ESI: pesticides, steroids and some lipid metabolites. APCI is robust against matrix effects and gives cleaner spectra than ESI for some compound classes; steroid panels at NDTL Delhi often run APCI for this reason.

Quantitation in LC-MS/MS follows a standardised procedure used across accredited toxicology laboratories. Spike every sample with a known concentration of the deuterated internal standard. Build a calibration curve from five to seven concentration points spanning the expected analyte range, run in the same matrix (blank urine, blank plasma) as the case sample. Fit the curve as analyte peak area divided by IS peak area versus analyte concentration, usually with a 1/x or 1/x² weighting because the calibration spans two to three orders of magnitude. Read the case sample's area ratio off the curve to get the concentration. LOQ for most drugs of abuse in this workflow sits between 0.1 and 1 ng/mL in biological matrix; LOD is typically a third of LOQ.

Data review is where analytical rigour is demonstrated. The analyst opens each chromatogram, checks the extracted ion chromatogram (XIC) for the quantifier and qualifier transitions, verifies that both peaks coelute, verifies the retention-time match against the calibrator (within ±2 percent in the SAMHSA convention), verifies the qualifier-to-quantifier ratio against the calibrator (within ±20 percent), verifies that the IS peak is present at the expected area, and verifies that the calibrator and QC samples have passed in the same batch. Anything that fails any check gets re-run or rejected.

The SAMHSA / SOFT confirmatory hierarchy is short:

1. Presumptive screen (immunoassay, UV-Vis, TLC, colour test) flags a positive
2. The same sample is split and prepared for confirmatory analysis
3. Confirmatory MS run with at least two transitions, deuterated IS, full calibration and QC
4. Both transitions present, retention-time match, ratio match, calibrator and QC pass
5. Report the quantified concentration with the technique, the LOQ, the analyst, the date and the chain-of-custody reference

Step 1 alone is never reported as a positive case finding. The phrase "presumptive positive, pending confirmation" is the standard wording. Cases successfully challenged at trial almost always failed at step 3 or step 4, not at step 1.

Toxicology instrumentation in India is distributed across three tiers. Every Tier-1 State Forensic Science Laboratory has a GC-MS. CFSL Chandigarh, CFSL Hyderabad, FSL Madhuban (Gujarat), FSL Kalina (Mumbai), FSL Madiwala (Karnataka), FSL Chennai and FSL Kolkata run Agilent 5977, Shimadzu QP2020 or Thermo ISQ instruments as their core toxicology workhorses. AIIMS Forensic Medicine in Delhi maintains its own GC-MS for autopsy-derived samples.

LC-MS/MS sits at a smaller set of centres. CFSL Chandigarh runs an Agilent 6470 triple-quadrupole as its confirmatory workhorse for drugs of abuse, prescription pharmaceuticals and emerging analytes. AIIMS Forensic Medicine runs an LC-MS/MS for clinical toxicology cases and forensic post-mortem work. The National Dope Testing Laboratory (NDTL) Delhi, India's WADA-accredited anti-doping laboratory, runs multiple LC-MS/MS systems for the steroid and prohibited-substance panels under WADA technical document specifications. A number of state SFSLs, including Hyderabad and Madhuban, have added LC-MS/MS over the last five years.

High-resolution MS in Orbitrap format is rare. NIPER Mohali runs an Orbitrap for pharmaceutical impurity profiling and pharmacokinetic studies. NCBS Bangalore runs Q-Exactive instruments (a hybrid quadrupole-Orbitrap platform) for metabolomics and lipidomics. NDTL Delhi has added a high-resolution Q-Exactive for anti-doping screening of emerging substances under the WADA prohibited list. The Centre for DNA Fingerprinting and Diagnostics (CDFD) Hyderabad and a few CSIR institutes hold additional units. Routine forensic toxicology cases rarely require these instruments; cases involving a suspected novel psychoactive substance or poisoning by an unknown plant alkaloid are referred to the appropriate research centre under a memorandum of understanding.