HYDROLYSIS, TISSUE ANALYSIS, HAIR,

DERIVATIZATION
Hydrolysis of conjugated metabolites
◦ Cleavage of conjugates is an important step in toxicological analysis, especially of
urine. Many drugs and metabolites (e.g. benzodiazepines, laxatives, opiates and
steroids) are excreted in urine and in bile predominantly as conjugates with D-
glucuronic acid or with sulfate, or sometimes with both.

◦ In order to maximize sensitivity in drug/poison screening, and, if appropriate, in order to

◦ All strong acid hydrolysis procedures are likely to introduce additional, hitherto unseen
compounds into sample extracts even if due care is taken to ensure effective
neutralization/buffering during sample preparation.
◦ In contrast, incubation with beta-glucuronidase and/or arylsulfatase (15 h, 35 ◦C) can
give selective hydrolysis of conjugates under relatively mild conditions.
◦ Generally cleaner extracts result, but the incubation time is longer (usually overnight),
there may be matrix effects from the enzyme solution ,and the enzyme preparations
are relatively expensive.
◦ There is a range of enzymes available. preparations of the digestive juice of Helix
pomatia, which contain both enzymes, are widely used. beta-Glucuronidase and
arylsulfatase from Patella vulgata are also marketed.
◦ The enzymes exhibit maximum activities at different pH values. The optimal pH for the
H. pomatia enzyme is pH 4.5 whilst that for E. coli is about 6.8. Furthermore, the H.
pomatia enzyme can be incubated at temperatures up to 45 ◦C, which can reduce
the incubation time markedly. Some temperature-tolerant preparations allow
incubation up to 60 ◦C (2 h incubation).
◦ Control experiments should be performed to ensure that the incubation conditions are
optimum with regard to the degree of hydrolysis and stability of the analytes.
◦ It should also be borne in mind that prolonged incubation of potentially infective
samples may increase the titer of the infective agent.
◦ Finally, note that beta-glucuronidase is specific for beta-D-glucuronides and some
glucuronides rearrange during incubation, and therefore cannot be hydrolyzed
enzymatically.
 Extraction of drugs from tissues
◦ Conventionally measurements in portions of organs such as liver and brain have been
performed via mechanical homogenization using a polytetrafluorethylene (PTFE)-in-glass
homogenizer, and/or acid digestion on, say,

◦ 5 g wet weight of tissue prior to solvent extraction at an appropriate pH. Other tissues such
as lung or muscle generally require use of a cutting or mincing action. However, digestion
(12–16 h, room temperature) of small amounts (say 100 mg) of tissue with collagenase,
protease, or lipase often gives much improved recovery when compared to conventional
procedures and has the advantage that, once the digest has been prepared, analogous
methodology and calibration standards to those used with plasma can be employed .

◦ It is obviously important to ensure that the enzymatic hydrolysis does not destroy the
analyte(s), hydrolyze metabolites to reform the analyte(s), or introduce other interferences.
An internal standard can be added to the homogenization buffer or at a later stage.
Hair analysis for drugs and organic
poisons
◦ Human hair consists of protein (65–95 %, mainly keratin), water (15–35 %) and lipid (1–9
%) together with some trace components including trace elements and heavy metals .
Drugs and metabolites are incorporated in the structure of hair as it is synthesized in the
hair follicle,

◦ but may also arise due to diffusion from skin and sebum, an oily secretion of the
sebaceous glands that helps to preserve the flexibility of hair. It is important to be able
to differentiate compounds incorporated into hair that arise from within the body from
substances that may contaminate the hair once it has grown away from the skin. Thus,
after measurement of the length of the hair sample and segmentation if required
(normally segments no less than 10 mm in length are used), most hair analysis protocols
incorporate a decontamination step prior to the actual analysis
◦ Pre-analytical
◦ Decontamination with detergents such as shampoos, surgical scrubbing solutions, surfactants [0.1 %
sodium dodecylsulfate (SDS) for example]; phosphate buffer; organic solvents such as acetone, diethyl
ether, methanol, ethanol, dichloromethane, hexane, or pentane (various volumes, various contact
times)
◦ Analysis of washings to monitor removal of any surface contamination
◦ Measurement of length from scalp/body end and segmentation if required
◦ Accurate weighing of dried hair segments and fragmentation (fine cutting, ball mill)
◦ Sample preparation
For immunoassay
◦ Incubation in aqueous buffer
For GC-MS, HPLC-MS, HPLC-MS-MS
◦ Incubation with internal standard solution, and
◦ Incubation in acidic or basic aqueous solution, and LLE, SPE, or SPME, with derivatization if necessary, or
◦ Incubation in organic solvent (often dichloromethane, methanol or acidified methanol) and LLE, SPE, or
SPME, with derivatization if necessary, or
◦ Enzyme digestion and LLE, SPE, or SPME, with derivatization if necessary
Assay calibration and reporting results
◦ Incubate ‘blank’ hair with known amounts of analyte across the anticipated concentration range
◦ Calculation of results (e.g. ug /g hair)
◦ Generally, a single washing step is used, although a second identical wash is sometimes performed.
◦ If external contamination is found by analyzing the initial washings, repeated washing/analysis cycles
may demonstrate that any surface contamination has been removed prior to the actual analysis.
◦ It has been suggested that the analyte concentration in the hair after washing should show a 10-fold
increase over the concentration in the last wash for a positive result to be accepted .
◦ After the washing stages, the dried hair or hair segments may be chopped finely with a razor blade or
pulverized in a ball mill prior to solubilization of drug and any metabolite(s) remaining in the hair.
◦ The choice of sample preparation procedure and the precise conditions used (such as the pH and the
molarity of aqueous incubation solutions, and the duration and temperature of incubation) depends on:
(i) the analyte(s) and
(ii) the analysis system to be used (RIA, GC- MS, etc.)
◦ If the sample is incubated in sodium hydroxide solution solubilization of the hair is complete, but
compounds such as cocaine and 6-monoacetylmorphine (6-MAM) will be hydrolyzed. Alternatively,
incubation in hydrochloric acid (0.1 mol L−1 for 16 h at room temperature, 45 ◦C, or 56 ◦C; 0.6 mol L−1 for
30 min at 120 ◦C) will minimize loss of such analytes

◦
◦ Use of enzymatic digestion with, for example, proteinase K, or beta-
glucuronidase/arylsulfatase , is a further option.
◦ Extraction with organic solvent has involved incubation in an ultrasound bath (45 ◦C, 6
h or so) and has the advantage that GC-MS or derivatization followed by GC-MS can
be carried out directly following solvent evaporation. Hydrolysis of labile analytes is
minimal. Use of HPLC-MS-MS can simplify sample preparation even further.
◦ Detection of drug metabolite(s) in hair that cannot be explained by surface
contamination or hydrolysis of surface contaminants provides evidence that positive
findings have arisen as a result of systemic exposure.
◦ Cocaethylene and norcocaine are examples, but there is still debate as to the
problems posed by external contamination, particularly for ‘crack’ (cocaine free
base), cannabis and heroin when smoked. It has been suggested that it is not possible
to remove contamination from such sources by conventional washing procedures .
Derivatization
◦ Generally derivatization should be avoided as it adds an extra step or steps to the analytical
procedure and thus may increase the possibility of error.
◦ This being said, in GC derivative formation is performed to achieve satisfactory
chromatography, to improve the detection characteristics of an analyte and sometimes to
provide additional evidence of compound identity.
◦ Derivatization may be used to shorten or lengthen the retention time as required, and also to
permit the separation
◦ In HPLC, derivative formation, although sometimes used to stabilize an analyte, is seldom
needed to achieve satisfactory chromatography except when performed to permit the
separation of enantiomers. Derivatization is thus used mainly to enhance the selectivity and
sensitivity of detection.
◦ The choice of reagent and hence the reaction conditions will be based on the functional
group to be derivatized, the presence of other functional or labile groups in the molecule,
and the reason for performing the derivatization. Generally, three types of reactions are
performed, viz.
◦ silylation, acylation and alkylation.
◦ In GC, silylation generally reduces retention, whereas acylation usually increases retention.
The order of elution for derivatives of a homologous series will be the same as the
underivatised parent compounds.