Methods for impurity profiling
17
Method A1:
High-performance liquid chromatography (HPLC) method*
Source: I. S. Lurie and S. M. Carr, “The quantitation of heroin and selected basic
impurities via reversed phase HPLC: I. The analysis of unadulterated heroin samples”,
Journal of Liquid Chromatography and Related Technologies, vol. 9, No. 11 (1986),
pp. 2485-2509.
Sample type: Major components: cut and uncut samples.
Operating conditions:
Detector:
Ultraviolet diode array
Monitor 3 wavelengths: 210 nm, 228 nm and 240 nm
Column:
Partisil 5, ODS 3, 125 mm x 3.2 mm ID
Mobile phases:
(a) Phosphate buffer (pH 2.2)
(b) Methanol
Injection solvent: HPLC-grade water, acetonitrile and glacial acetic acid
(89:10:1), adjusted to pH 3.7 with 2M sodium hydroxide
Flow rate:
0.76 ml/min
Injection size:
20 µl
Elution gradient:
Equilibration
Time
MeOH (%)
Buffer (%)
15
5
95
1
20
30
70
2
6
30
70
3
10
80
20
4
4
80
20
5
5
5
95
Internal standard: propiophenone at 0.5 mg/ml
in injection solvent
Phosphate buffer: 870 ml of HPLC-grade water, 30 ml of 2N sodium hydroxide and
10 ml of phosphoric acid. Filter and degas, then add 3.0 ml hexylamine. The final
pH is adjusted to pH 2.2 with 2M sodium hydroxide or phosphoric acid. Add addi-
tional hexylamine as necessary for baseline separation of papaverine and noscapine.
Sample preparation: Accurately weigh the sample into a 100-ml
volumetric flask to
give an approximate heroin concentration of 0.9 mg/ml. Add 10 ml of propiophenone
internal standard solution and dilute to volume with injection solvent. Sonicate to com-
plete solvation and filter.
Rationale for use: A robust method providing accurate quantification and excellent
precision for heroin and all typical opium alkaloid impurities down to 1% relative to
heroin content. In many cases minor alkaloids and adulterants can be quantified at
*This HPLC method is a slightly modified version of the “classic” heroin signature 1 method
used by the Drug Enforcement Administration (DEA) of the United States of America in its heroin
origin determination programme, until it was replaced in 2003 by a capillary electrophoresis (CE)
method (method A6).
18
Methods for impurity profiling of heroin and cocaine
Method A2:
GC method, without derivatization
Source: Modified from C. Barnfield and others, “The routine profiling of forensic
heroin samples”, Forensic Science International, vol. 39, No. 2 (1988), pp. 107-117.
Sample type: Major components: cut and uncut samples.
Operating conditions:
Detector:
Flame ionization detector (FID)
Column:
DB-1, PB1 or equivalent, 25 m x 0.32 mm x 0.5 µm
Carrier gas:
Helium
Make-up gas:
Not specified
Injection:
1 µl; split 30:1
Temperatures:
Injector: 280°
C
Detector: 320°
C
Oven:
200° C to 260° C at 10° C/min, to 310° C
at 30° C/min, hold for 1 min
Internal standard: None: normalize all data relative to heroin response.
Sample preparation: Weigh out the powder and dissolve initially in one part of
N,
N-dimethylformamide,* then dilute with nine parts of ethanol to give a final concen-
tration of 3-5 mg/ml of sample, depending on the heroin concentration.
levels as low as 0.1% relative to heroin content along with significant but generally
acceptable losses in precision. The minor alkaloids quantified are morphine, codeine,
O3MAM, O6MAM, papaverine and noscapine. As is the case with every analytical
procedure for heroin, hydrolysis can be an issue, but if the method is followed pro-
perly, hydrolysis will be kept to an absolute minimum. The original reference states:
“Quantitative values of the various basic impurities relative to heroin in the samples
analysed were found to vary over a large range and formed a basis for comparing
illicit heroin samples.” The on-column heroin content specified in this method typi-
cally produces an UV-detector response that is in the upper region of detector linear-
ity. Hence, it is necessary for the analyst to obtain a “rough” quantification of the
heroin prior to analysis by this method. Any of the following GC methods designed
for major impurity analyses can be utilized for this purpose. The use of a photo-diode
array (PDA) detector is recommended as it allows the facile detection of co-eluting
compounds, that is, peak purity assessments. Precision and accuracy for morphine are
somewhat limited, as morphine elutes very soon after column void volume. Common
co-elution issues occur for acetaminophen (paracetamol) with codeine, cocaine with
acetylcodeine and diphenhydramine with noscapine. Sugars are not detected.
Outcome: Indication of general source region (South-East Asia, South-West Asia,
Mexico and South America). Sample comparisons for discrimination and evaluation
of samples for case-to-case evidential purposes (linkage determinations). Additional
information is required to confirm links between samples or to assign source regions,
that is, the method should be used as one part within a broader analysis scheme.
*The use of N,N-dimethylformamide facilitates the dissolution of samples containing large
proportions of caffeine, phenacetin and/or paracetamol.