This invention relates to a process for the quantitative determination
of cocaine and other alkaloids, such as morphine and methadone, in a solid sample,
e.g., a sample of hair, by using a screening type approach.
The invention also relates to a reagent for use in such process and
new diagnostic kits including such reagent among their components.
By "screening type approach" is meant a kind of analysis permitting
to analyze in a relatively short time span a relatively large number of samples
in a cheap, efficacious and standardized manner. This kind of analysis permits
to exclude the negative samples by immediately identifying the samples that do
not contain the substance or the entire substance class or those in which said
substances are present at a level lower than the threshold or cut-off value.
The threshold or cut-off is a practical limit selected to establish
if the sample analyzed is positive or negative. The threshold value differs from
the limit of detection of the method, that is, the lowest concentration of an analyte
that can be determined. In fact, the cut-off value is normally set at a concentration
higher than the limit of detection in order to obviate the imprecision of the
analysis at values close to the limit of detection.
Cut-off values are conventionally established and take into account
a multiplicity of factors, such as the capability of using commercially available
reagents, the pharmacokinetic properties of the substances and the need to avoid
false negatives (see: P.Zuccaro, S.Pichini, I.Altieri and R.Pacifici: Proposta
di linee guida per l'analisi delle sostanze d'abuso nei liquidi biologici The substances
that the metabolism of the human body accumulates in the hair are numerous. Usually,
it is required to detect the presence of alkaloids and other substances of abuse,
such as, morphine, methadone and/or cocaine. By the method of this invention further
substances can also be detected, such as, those of the following non-limitative
list: 6-O- mono-acetyl-morphine, bi-acetyl-morphine, codeine, papaverine, nalorphine,
nicotine, cotinine, caffeine, noscarpine, mepivacaine, trimetropin, buprenorphine,
pentazocyne, methadone metabolyte, benzoylecgonine, amphetamine, metamphetamine,
methylenedioxyamphetamine, methylenedioxymetamphetamine, methylenedioxyethylamphetamine,
Description of the state of the art
Several techniques are known for the determination of the analytes
of interest, such as those mentioned above. Specifically, for the analysis of
cocaine that is present in hair, gas chromatography (GC) combined with Mass Spectrometry
(MS) hereinafter referred to as GC/MS and Radio Immune Assay technique (R.I.A.)
GC or liquid chromatography (LC) combined with MS are methods of
resolution, purification or separation and identification of components of complex
mixtures of organic or inorganic substances having even strictly similar chemical
properties. The separation of substances dissolved in a liquid or fixed on a finely
divided or porous solid substance is based on percolation, respectively elution
trough them of an eluent gas, respectively liquid.
When the substances to separate/detect can be made gaseous and as
eluent a gas is used, GC applies. The latter is a separation method based on the
distribution between a solid or liquid stationary phase and a mobile phase made
of the gases or vapors to separate, which are carried by a stream of an inert gas.
The analytical results are reported in a graph named chromatogram,
in which the quantities of the single components present in the mixture and transferred
to the eluent gas/liquid are reported versus time. The graph has peaks whose highness
is a direct function of the quantity of the specific substance.
Chromatographic methods that can be used for confirmatory analyses
are GC and LC, the latter being often referred to as High Performance Liquid Chromatography
(HPLC). The most commonly employed detectors for GC are electron scatting detectors
and phosphor nitrogen detectors.
As said above, GC/MS employs a gas chromatograph coupled to a mass
spectrograph. By so doing, the separation capability of GC combines with the specificity
proper of MS. Therefore, GC/MS represents the method of choice for confirmatory
analyses of the above named substances as well as their metabolites (see again
"Proposta di linee guida per l'analisi delle sostanze d'abuso nei liquidi biologici"
by ISTITUTO SUPERIORE DI SANITA')
In the conventional acid hydrolysis, however, cocaine is extracted
by hydrochloric acid as it is, i.e. without undergoing transformation into its
metabolite benzoylecgonine. Since the amounts of cocaine extracted in this way
are very little, it is not possible to determine cocaine by the usual screening
Radio Immune Assay (RIA) is based on radio-immunological tests using
known amounts of antibodies and of analyte labeled with a radioisotope (generally
I125). During incubation the labeled analyte and that possibly present
in the sample compete for the antibody sites. After precipitation of antigen-antibody
complexes and centrifugation the supernatant or the precipitate are transferred
to a gamma Geiger counter that measures the radioactivity level. RIA kits are extremely
sensitive and allow identification of 1-5 ng of substance per ml. Adoption of automatic
instruments for pipetting and counting allows the contemporaneous analysis of numerous
samples with response times of 1 to 5 hours. On the other hand, the use of radioactive
isotopes requires adequate safety measures. Furthermore, the relatively short
half-life of the radioactive isotopes imposes a careful handling of reagents.
RIA does not lend itself to a widespread use for the quick determination
of the substances in question in view, first of all, of the high cost of reagents,
further increased by liability of the antisera. Secondly, in view of the criticality
of the analysis due to the need of operating with radioactive materials that are
dangerous for the analysts and also in view of the required times which are relatively
long. RIA also requires the availability of rooms properly equipped and shielded,
as well as particular care in handling waste materials and in their disposal.
Therefore, RIA is scarcely employed in this sort of analyses. The
prior art shows therefore the impracticality of determining cocaine present in
the hair by the so-called screening type approach, as described above, i.e., by
very quick and cheap techniques as are used for the determination of the same
substances in urine. This is mainly due to the fact that in the conventional screening-type
approach what is analyzed is not cocaine, but its metabolite benzoylecgonine. The
fact that this substance does not exist in nature by itself, but only as the metabolyte
of cocaine and the fact that the reaction of transformation of cocaine into benzoylecgonine,
i.e. the transformation of the ester group into an hydroxyl group, is irreversible
in an alkaline environment render the determination of benzoylecgonine a necessary
and sufficient condition for the detection of cocaine.
Benzoylecgonine is found in the hair in a ratio of 1 to 4 or even
1 to 10 and more with respect to cocaine. Therefore, when the amount of cocaine
is very low or close to the cut-off values (0.2-0.1 ng cocaine per mg hair) screening
cannot be performed because of the low amount of this metabolyte (0.05-0.025 ng
benzoylecgonine per mg hair), such amount being undetectable by the screening apparatuses
available in the laboratories.
US-A-5 910 419 which is the nearest prior art to the invention relates
to a method for screening hair samples using the well known ELISA technique for
the presence of cannabinoids and further RIA for the presence of cocaine. As already
outlined, the radio immunoassay does not lend itself to quick and cheap determination
of drug of abuse and further involves safety and handling difficulties.
Summary of the invention
The main object of this invention is to overcome the above mentioned
drawbacks, i.e. the impossibility to dose the cocaine present in a solid sample
by the conventional screening-type approach.
In accordance with the invention, this object is achieved by a screening-type
procedure for the quantitative determination of cocaine and other alkaloids which
are present in a solid sample which, in accordance with claim 1, includes the following
- a) preparing a solid sample in a finely divided or powdered form;
- b) selecting a liquid reagent providing constant concentration of hydroxyl
groups suitable for extracting and transforming cocaine into benzoylecgonine and
for extracting other similar substances;
- c) extracting cocaine and other similar substances contained in the sample
and transforming the extracted cocaine into benzoylecgonine by maintaining the
sample completely immersed in said liquid reagent at a temperature ranging from
10°C to 250°C for a period of time ranging from few seconds to 48 hours; and
- d) analysing the liquid separated from the solid sample to determine the concentration
of benzoylecgonine contained in said liquid with respect to the cut-off limit using
a conventional screening kit for the determination of the said substance in urine.
Preferred aspects of the invention include using hair as the solid
material forming the solid sample; using a buffer as the reagent which provides
hydroxyl groups, more preferably an ammonia buffer, most preferably a buffer that
is hereinafter referred to as VMA; and heating the sample immersed in the liquid
at a temperature of about 100 to 150°C for about one hour.
In accordance with another aspect of the invention a process is provided
which includes the additional steps of:
- arranging samples by increasing concentrations of the substances of interest;
- performing confirmation analyses with known techniques, such as, GC or GC/MS.
In one embodiment of the invention, the process may provide the following
- providing a sample made of about 50 to 300 mg of finely divided and/or powdered
- adding in the test tube containing the said sample a suitable liquid reagent
until the sample is completely immersed, said reagent being capable of performing
extraction and transformation of cocaine into benzoylecgonine and at the same time
of extracting other similar substances which are present in the sample
- if necessary, agitating the test tube to facilitate immersion of the sample;
- heating the contents of the test tube to a temperature T1 for a time interval
t1 by keeping the test tube immersed in a thermostated bath or by placing it in
- cooling the test tube to room temperature;
- taking the liquid and transferring it into a test tube suitable for a screening
- performing the screening by using a kit of reagents for the determination of
the said substances in urine;
- reading the data resulting from the first level instrumentation to verify the
concentration values with respect to the cut-off limit; and
- contemporaneously determining the amount(s) of substance(s) present.
In accordance with another aspect of the invention a reagent is provided
for use in the above mentioned process which-in the most -preferred embodiment
has the following formula:
VMA = 0.2 M (NH4)2HPO4 + 5ml/L 25% NH4OH
In the above formula (NH4)2HPO4 is
dibasic ammonium phosphate and NH4OH is ammonium hydrate. In fact, 5ml/L
NH4OH give a 0.07 M concentration of hydroxyl groups, which is comprised
in the range giving 100% conversion.
In addition to the main advantage of the invention as outlined above,
another advantage is that the confirmation analyses (e.g., GC/MS) need to be performed
only on those samples that have been determined to be positive at the initial screening
whereas, when conventional techniques are used, all samples must be analyzed since
there is no kit available for determination of cocaine, but only for determination
In average, the analysis of 25 samples requires two hours for the
preparation of the samples plus one week for the confirmation analyses of all (both
positive and negative) samples.
On the other hand, by applying the present invention, an analyst will
require for the same 25 samples two hours for samples preparation plus 30 minutes
for the screening-type analysis plus the time necessary for performing the confirmation
analyses. However, the latter need to be performed only on those samples which
have been determined to be positive.
Since, in the average, positive samples are between 0 and 20% and
since most of the time required is spent in the confirmation analyses, the procedure
according to the present invention allows saving of substantial time which is
estimated at about 70 to 90% of the time required for the analysis of the 25 samples
taken into consideration.
Another advantage according to this invention is that the possible
dragging of the active substances from one sample to another in the confirmation
analyses is eliminated or minimized because it is possible to arrange samples
in order of increasing concentration of the substances of interest, therefore proceeding
to confirmation analyses starting from those samples having the lowest concentration
and then with those having higher and higher concentrations.
In this way, it is completely eliminated the possibility that a sample
with high concentration of, e.g. cocaine, leaves a trace of it in the instrument
used for the analysis and has an impact on the determination of the same substance
in the following sample, perhaps having a concentration just below the cut-off.
Still another advantage of this invention is that it allows to search
and determine at the same time and in the same sample not only cocaine but also
other substances, such as, morphine by applying the same cut-off limit or else
methadone by modifying the limit of cut-off.
One further advantage is that the procedure according to this invention
offers a higher guarantee to the analyst because positive outcome is confirmed
by two different analytical methods.
In a further aspect of the invention a diagnostic kit is provided
including the reagent described above as one of its components.
Still further additional advantages will appear from the reading of
the following detailed description and the following non-limitative examples.
Brief Description of the Drawings
Detailed description of invention embodiments
- Figure 1 is a chromatogram obtained from an apparatus performing gas chromatography
on the sample of Table 2;
- Figure 2 is the graph obtained by Mass Spectrometry on the first sample after
acid hydrolysis and shows presence of cocaine;
- Figure 3 is the Mass Spectrum of the same first sample after treatment according
to the present invention and shows presence of benzoylecgonine;
- Figure 4 is the Mass Spectrum of the same first sample after treatment according
to the present invention and shows presence of morphine;
- Figure 5 is a chromatogram similar to that of Figure 1 and refers to the first
sample treated according to this invention as shown in Table 3;
- Figure 6 is similar to the MS of Figure 3 showing presence of benzoylecgonine;
- Figure 7 is similar to the MS of Figure 3 showing presence of morphine;
- Figure 8 is similar to Figure 1 but relates to a second sample;
- Figure 9 is similar to Figure 5 but relates to a second sample;
- Figures 10 and 11 are respectively similar to Figures 8 and 9 but relate to
a third sample;
- Figures 12 and 13 are respectively similar to Figures 8 and 9 but relate to
a fourth sample;
- Figure 14 is a diagram showing transformation of cocaine into its metabolite
- Figure 15 is a graph showing concentration of cocaine versus concentration of
OH- when the reaction of transformation of cocaine into benzoylecgonine
occurs at 100° C for 1 hour; and
- Figure 16 is similar to Figure 15 and shows the case in which the reaction
occurs at 150°C for 1 hour.
The process of this invention substantially is a process for the quantitative
extraction and transformation of cocaine into benzoylecgonine and for the extraction
of similar alkaloids, in particular toxic substances of abuse and/or drugs, which
are present in a sample, prepared starting from a solid material.
In the following detailed example, the sample is obtained starting
from finely divided hair.
The procedure allows, in particular, the dosage of cocaine by a screening
type technique, at the same time allowing the dosage of possible other toxic substances
and the like present in the hair, by using kits of reagents available in commerce
and intended for the analysis in urine. The following steps are carried out:
- crush hair in fragments of two-three mm length;
- weigh the fragments to form a sample having a weight of 50 to 300 mg;
- wash the sample with methanol in a closed test tube at room temperature to
eliminate possible external substances that might interfere with the results, such
as, for example, traces of drugs external to the hair that have deposited on it
because of its presence in the air (by so doing, it is possible to distinguish
if the hair was that of a handler or that of the consumer of the drug);
- Repeat the step of washing with methanol, if necessary;
- wash the sample with ethanol in a closed test tube to eliminate traces of methanol
- dry in an oven at about 45°C under flow of inert gas, such as, nitrogen;
- add into the test tube 0.5 to 2 ml of the reagent as defined above and shake
- heat the test tube to 100°C and maintain at this temperature for 1 hour, e.g.
by means of a thermostatic bath or an oven;
- cool the test tube to room temperature, e.g., by immersing the tube into cold
water or simply leaving it at room temperature for a suitable time span;
- take the liquid and pour into a test tube of the kind used for urine examination
(if necessary, centrifuge the test tube to eliminate turbidity);
- insert the tube in a "first-level screening apparatus" for the quantitative
determination of the substances sought for (benzoylecgonine, morphine, methadone,
- adjust settings of the first-level apparatus in a way suitable for small amounts
(this may be necessary if the apparatus is alternatively used for determination
in hair or urine);
- perform screening -type analyses using the reagents provided with the kit normally
employed for the examination of urine;
- read data resulting from the first-level analyses and establish positivity or
negativity with reference to the cut-off value.
The above procedure may then be complemented with the following additional
steps when confirmation analyses are required:
- arrange the samples in the order of increasing concentration of the sought
for substances, determined as above described;
- perform confirmation analyses by analyzing the samples taken in the order of
the arrangement (In this way the above mentioned problems of dragging traces of
drugs from one sample to the other are overcome).
The invention process provides for the use of a reagent (hereinafter
referred to as VMA) which is a buffer solution. The buffer serves for transforming
the cocaine present in the hair into its metabolite benzoylecgonine, as shown in
the scheme of Figure 14. Cocaine, in the presence of hydroxyl groups and at a
suitable temperature first is transformed into an intermediate product and then
into benzoylecgonine plus methanol.
Buffer solutions are obtained by reacting a salt with its weak base.
These solutions have a stable pH; therefore the VMA reagent is able to produce
hydroxyl groups in a steady way. The use of solutions in which the production
of hydroxyl groups is not regular creates problems when the cocaine concentration
is close to the cut-off limit.
As said above, the composition of the buffer for use in the process
is preferably the following:
VMA = 0.2 M (NH4)2HPO4 + 5ml/L 25% NH4OH
Alternatively, VMA may be replaced by solution in which the component
being the source of hydroxyl groups is selected among the following non-limitative
list of substances: aluminum hydroxide, barium hydroxide octahydrate, benzyltriethylammonium
hydroxide, benzyltrimethylammonium hydroxide, calcium hydroxide, lithium hydroxide,
lithium hydroxide monohydrate, magnesium hydroxide, potassium hydroxide, potassium
hydroxyantimoniate, sodium hydroxide, sodium hydroxide monohydrate, strontium hydroxide
octahydrate, tetramethylammonium hydroxide, tetrapropylammonium hydroxide, trimethylvinylammonium
As solvent, any of the following can be used in alternative:
- ethanol, methanol, water, monobasic ammonium phosphate, ammonium acetate, ammonium
benzoate, ammonium bicarbonate, ammonium bichromate, ammonium bisulphate, ammonium
bromide, ammonium carbamate, ammonium carbonate, ammonium citrate bibasic, ammonium
chromate, ammonium iodide, molibdate, ammonium monovanadate, ammonium nitrate,
ammonium oxalate monohydrate, ammonium persulphate, ammonium sulphate, ammonium
sulphamate, ammonium sulphite, ammonium sulphide, ammonium tartrate, ammonium thiocyanate,
ammonium thioglycolate, ammonium thiosulphate, ammonium chloride, sodium phosphate
monobasic, sodium phosphate bibasic, potassium phosphate monobasic, potassium
Figure 15 shows a graph in which the percentage of cocaine transformed
into benzoylecgonine is reported versus the concentration of OH- when
the reaction is carried out at the temperature of 100°C for 1 hour. From the graph
it appears evident that the transformation is maintained at high levels (at least
70%) for a range of OH- concentrations of from 0.03 to 0.5 M.
From the graph of Figure 16, which shows hydroxyl concentrations after,
respectively, 0, 15, 30 and 60 minutes in a reaction carried out at 150°C; it
appears evident that the percentage of transformed cocaine is high only around
the abscissa point of 15 minutes.
From all of the above and numerous other experiments the optimal values
for the reaction temperature and time result to be, respectively, 100°C and 1
The examples reported below show the quantitative determination of
cocaine in hair performed both with known techniques and with the process of the
invention. Analyses have been carried out using the following instruments: for
the screening analyses, the ROCHE instrumentation named "COBAS MIRA PLUS" which
uses reagents provided by the same company and named "ABUSCREEN ON LINE"; for GC/MS
the instrument provided by the company VARIAN which is named "SATURN GC/MS", model
Before proceeding to the various screening analyses the apparatus
has been controlled with a sample positive to both cocaine and morphine in order
to verify feasibility of the methodology.
As can be seen from Table 1 the sample resulted positive to both cocaine
The concentration values detected by the apparatus resulted to be
0.16 for morphine and 0.15 for cocaine, in line with the expected values for both
substances present in the control sample.
In this example a sample has been analyzed which resulted, in the
end, to be positive to both cocaine and morphine.
The sample was subjected to conventional analysis with acid hydrolysis
and then subjected to screening analysis which gave the following results:
As can be seen, acid hydrolysis was able to extract an amount of morphine
higher than the limit, but the amount of cocaine resulted to be too little to
allow detection by the screening apparatus. The reason is that cocaine was extracted
as such and not transformed into its metabolite benzoylecgonine, which latter is
just what present screening methods detect.
The same sample was then subjected to confirmatory analysis by GC/MS
with the results reported in Figures 1 to 4. Specifically, from Figure 1 it can
be seen that the chromatogram of the first sample shows a high peak at the position
of cocaine and a very small peak at the position of benzoylecgonine.
The same Figure 1 also shows confirmation of the presence of morphine.
Additional confirmations of the presence of both cocaine and morphine
result from the graphs of Figures 2, 3 and 4.
The sample of Example 1 was then subjected to the procedure of this
invention to give the results shown in Table 3.
It is immediately clear that the values detected for both morphine
and cocaine are higher than the respective limits, therefore the sample is positive
The high value detected for cocaine is to be ascribed to the benzoylecgonine
that has been produced during the transformation reaction.
The same sample of Example 1 treated with the process of this invention
has been subjected to GC/MS confirmation analyses with the results reported in
Figures 5 to 7. As can be seen from Figure 5, the cocaine peak practically cannot
be seen anymore because cocaine has been completely transformed in benzoylecgonine,
the peak of which is well apparent in the same Figure 5.
Again in this example a sample which, in the end, resulted to be positive
to both cocaine and morphine has been analyzed first by the conventional analytical
method of acid hydrolysis and screening with the results reported in Table 4.
Like in the preceding Example, acid hydrolysis was able to extract
an amount of morphine higher than the limit, but the quantity of cocaine extracted
was too little to allow detection by the apparatus.
The sample was then subjected to GC/MS confirmatory analyses to give
the results reported in Figure 8 where it can be seen that the chromatogram shows
a high peak at the position of cocaine and a negligible peak at the position of
The sample of Example 2 was then subjected to the invention process
and to screening analyses with the results reported in Table 5.
Both the cocaine and the morphine values are higher than the respective
limits, therefore the sample is positive to both.
The sample of this Example, treated according to the invention process,
was also subjected to GC/MS confirmation analyses with the results reported in
Figure 9 where it can be seen that the cocaine peak is not practically present
anymore whereas the benzoylecgonine peak is high.
In this Example a sample has been analyzed which, at the end, resulted
to be positive to cocaine only and not to morphine.
This sample was first subjected to acid hydrolysis and screening by
the conventional methods to give the results reported in Table 6.
By acid hydrolysis a quantity of morphine lower than the limit was
extracted. The quantity of cocaine was also too little to allow the equipment to
detect it. By using the methods known in the art, therefore, nothing can be said
as to the positivity or negativity of the sample and it was necessary to make
recourse to the GC/MS confirmation analyses that gave the results reported in Figure
10. In such Figure, the chromatogram of the sample of this Example shows a high
peak at the position of cocaine and a negligible peak at the position of benzoylecgonine.
This confirms once more that acid hydrolysis extracted cocaine as such without
The same sample of this Example has also been treated according to
the invention process and subjected to screening analysis. Results are reported
in Table 7.
It is immediately evident that while the value of morphine is lower
than the limit, that of cocaine is slightly higher than the cut-off and therefore
the sample is only positive to cocaine.
This same sample, after treatment with the invention process, has
been subjected to GC/MS confirmation analyses with the results shown in Figure
11. In this Figure, the peak of cocaine has practically disappeared because the
same was completely transformed into benzoylecgonine, the peak of which is, instead,
very high and well apparent.
This fourth Example also analyzes a sample which, at the end, resulted
positive to cocaine and not to morphine.
Analyses were carried out by applying the same scheme as in the preceding
Table 8 shows that upon analysis with acid hydrolysis the sample seems
to be completely negative to both morphine and cocaine, whereas Figure 12 indicates
the presence of cocaine in an amount higher than the limit.
The same sample, treated with the invention process (VMA reagent)
already on the first screening analysis shows a presence of cocaine higher than
the limit, see Table 9.
As usual, this result was confirmed by GC/MS analysis as shown in
Figure 13. The big peak of benzoylecgonine indicates that, before the treatment,
the sample contained cocaine in a percentage higher than the cut-off.