Forensically acceptable determinations of gestational fetal exposure to drugs and other chemical agents

A method for making a qualitative determination of maternal use of or exposure to at least one target analyte during pregnancy and related exposure of a subject infant to said at least one target analyte as a fetus, said at least one target analyte is selected from the group consisting of amphetamine, opiate, cocaine metabolite and cannabinoids, a qualitative determination of the presence or absence of said at least one target analyte is made in a sample of neonatal meconium obtained from said subject infant, said method comprising;

• providing a sample of neonatal meconium obtained from a newborn infant suspected of being exposed to said at least one target analyte in utero;
• contacting said sample with a substantially non-aqueous, volatile organic acid to form a first mixture said first mixture for a time sufficient to extract substantially all of said at least one target analyte from said meconium sample until a substantially uniform liquid dispersion is obtained;
• adding a substantially non-aqueous volatile organic solvent to said liquid dispersion to form a two-phase mixture including an organic phase and a second phase;
• thoroughly mixing said two-phase mixture for a time sufficient to permit substantially all of said extracted at least one target analyte, if present, to be extracted and collected in said organic phase;
• separating said organic phase from said second phase to form a substantially non-aqueous concentrated meconium extract for testing;
• and
• thereafter, analyzing said extract for the presence of said at least one target analyte.

A method for making a forensically acceptable determination of maternal use of or exposure to at least one target analyte during pregnancy and related exposure of a subject infant to said at least one target substance as a fetus in utero, said at least one target analyte is selected from the group consisting of amphetamine, cocaine metabolite, opiate and cannabinoids, said method comprising:

• (a) providing a first aliquot portion of a time-averaged pooled, pre-mixed meconium tissue sample for the subject infant;
• (b) contacting said time-averaged tissue sample with a substantially non-aqueous volatile organic acid to form a first mixture and agitating said first mixture for a time sufficient to extract substantially all of said at least one target analyte from the tissue sample and until a substantially uniform liquid dispersion is obtained;
• (c) adding a substantially non-aqueous volatile organic solvent to said liquid dispersion to form a two-phase mixture including an organic phase and a second phase;
• (d) thoroughly mixing said two-phase mixture for a time sufficient to permit substantially all of said extracted at least one target analyte, if present, to be extracted and collected in said organic phase;
• (e) separating said organic phase from said second phase to provide a substantially non-aqueous concentrated cocktail extract;
• (f) analyzing at least one aliquot portion of said cocktail extract in a preliminary screening immunoassay specific for said at least one target analyte to determine whether or not the at least one target analyte is present in said cocktail extract;
• and
• (g) if a positive result, indicating the presence of said at least one target analyte in said cocktail extract is obtained in step (f), then and thereafter;
• (h) providing a second aliquot portion of said time-averaged meconium tissue sample for the subject infant as in step (a), said second aliquot portion having a known weight;
• (i) spiking the second aliquot portion with a known amount of at least one internal control, said internal control being a labelled analog for said positively detected target analyte;
• (j) treating said spiked second aliquot portion to release bound target analyte and said added analogs into a distinct separatable phase;
• (k) separating said separatable phase to provide a second extract containing said released target analyte and added analogs;
• (l) analyzing said second extract by GC/MS methods to obtain an identifiable response curve associated with said target analyte and said analog;
• and
• (m) thereafter, quantitatively determining the amount of target analyte present in said second aliquot portion in terms of nanograms of target analyte per gram of meconium tested by multiplying the ratio of a response area of target analyte to a response area of the known analog times the amount of analog added divided by the extraction mass tested.

A method for making a forensically-acceptable quantitative determination of the concentration of amphetamine and methamphetamine target analytes present in a meconium sample, said method comprising:

• collecting and pooling quantities of newborn meconium from a subject infant during the postpartum period;
• thoroughly mixing the pooled meconium until a substantially uniform, non-striated smooth pre-processed meconium product is obtained;
• providing a test specimen comprising an aliquot portion of said pre-processed meconium product having a known weight;
• spiking said test specimen with internal controls by adding known amounts of deuterated drug analogs d-amphetamine-D.sub.5 and methamphetamine-D.sub.8 to said test specimen;
• adding an amount of a concentrated aqueous mineral acid sufficient to liberate said amphetamine target analytes and said spiked analyte analogs into an aqueous acid phase;
• separating said aqueous acid phase from a second phase containing meconium solids to form a first acidic extract;
• subjecting said first acidic extract to additional solvent extraction steps to selectively further separate target amphetamine analytes and analogs present in said first acidic extract from other undesired components such that said separated target amphetamine analytes and analogs end up in organic soluble form in a concentrated organic solvent solution;
• derivatizing said target analytes and analyte analogs present in said concentrated organic solvent solution to a derivatized form appropriate for gas chromatography mass spectrometric analysis;
• performing a GC/mass spectral analysis on said concentrated derivatized sample solution;
• and
• analyzing the results to quantitatively determine the amount of each said target analyte present in the meconium test specimen in terms of nanograms of amphetamine or methamphetamine analyte per gram of test specimen meconium by determining and multiplying the ratio of a response area of target analyte to a response area of the known analyte analog times the known quantity of the analyte analog added, divided by the mass of meconium test specimen.

A method for making a forensically-acceptable quantitative determination of the concentration of benzoylecgonine target analyte present in a meconium sample, said method comprising:

• collecting and pooling quantities of newborn meconium from a subject infant during the postpartum period;
• thoroughly mixing the pooled meconium until a substantially uniform, non-striated smooth pre-processed meconium product is obtained;
• providing a test specimen comprising an aliquot portion of said pre-processed meconium product having a known weight;
• spiking said test specimen with internal controls by adding known amounts of deuterated drug analogs and benzoylecgonine-D.sub.3 to said test specimen;
• adding a substantially non-aqueous, volatile organic acid to said test specimen to form a first mixture;
• agitating the first mixture for a time sufficient to release substantially all of said benzoylecgonine target analyte from the meconium sample, without diluting the sample with water, until a substantially uniform first liquid homogenate is obtained;
• adding a volatile organic solvent to said first liquid homogenate to form a two-phase mixture including an organic phase and a second phase;
• thoroughly mixing the two-phase mixture for a time sufficient to permit substantially all of said released benzoylecgonine target analyte and the analog to be extracted and collected into said organic phase;
• separating the organic phase from the second phase to provide a first organic extract;
• subjecting said first organic extract to additional solvent extraction steps to selectively further separate target and benzoylecgonine analyte and analog present in said first organic extract from other undesired components such that said separated target and benzoylecgonine analyte and analog end up in organic soluble form in a concentrated organic solvent solution;
• derivatizing said target analyte and analyte analog present in said concentrated organic solvent solution to a derivatized form appropriate for gas chromatography/mass spectrometric analysis;
• performing a GC/mass spectral analysis on said concentrated derivatized sample solution;
• and
• analyzing the results to quantitatively determine the amount of each said target analyte present in the meconium test specimen in terms of nanograms of benzoylecgonine analyte per gram of test specimen meconium by determining and multiplying the ratio of a response area of target analyte to a response area of the known analyte analog times the known quantity of the analyte analog added, divided by the mass of meconium test specimen.

A method for making a forensically-acceptable quantitative determination of the concentration of opiate morphine and codeine target analytes present in a meconium sample, said method comprising:

• collecting and pooling quantities of newborn meconium from a subject infant during the postpartum period;
• thoroughly mixing the pooled meconium until a substantially uniform, non-striated smooth pre-processed meconium product is obtained;
• providing a test specimen comprising an aliquot portion of said pre-processed meconium product having a known weight;
• spiking said test specimen with internal controls by adding known amounts of deuterated drug analogs codeine-D.sub.3 and morphine-D.sub.3 to said test specimen;
• adding an amount of a concentrated aqueous mineral acid sufficient to liberate said opiate target analytes and said spiked analyte analogs into an aqueous acid phase;
• separating said aqueous acid phase from a second phase containing meconium solids to form a first acidic extract;
• subjecting said first acidic extract to additional solvent extraction steps to selectively further separate target opiate analytes and analogs present in said first acidic extract from other undesired components such that said separated target opiate analytes and analogs end up in organic soluble form in a concentrated organic solvent solution;
• derivatizing said target analytes and analyte analogs present in said concentrated organic solvent solution to a derivatized form appropriate for gas chromatography/mass spectrometric (GC/MS) analysis;
• performing a GC/MS analysis on said concentrated derivatized sample solution;
• and
• analyzing the results to quantitatively determine the amount of each said target analyte present in the meconium test specimen in terms of nanograms of codeine and morphine analyte per gram of test specimen meconium by determining and multiplying the ratio of a response area of that target analyte to a response area of the known analyte analog times the known quantity of the analyte analog added, divided by the mass of meconium test specimen.

A method for making a forensically-acceptable quantitative determination of the concentration of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) target analyte present in a meconium sample, said method comprising:

• collecting and pooling quantities of newborn meconium from a subject infant during the postpartum period;
• thoroughly mixing the pooled meconium until a substantially uniform, non-striated smooth pre-processed meconium product is obtained;
• providing a test specimen comprising an aliquot portion of said pre-processed meconium product having a known weight;
• spiking said test specimen with internal controls by adding known amounts of deuterated drug analog THC-COOH-D.sub.3 to said test specimen;
• adding an amount of an alkanol solvent sufficient to liberate said target analyte and said spiked analyte analog into said alkanol phase;
• separating said alkanol phase from a second phase containing meconium solids to form a first alkanol extract;
• subjecting said first alkanol extract to additional solvent extraction steps to selectively further separate target THC-COOH analytes and analogs present in said first alkanol extract from other undesired components such that said separated target analytes and analogs end up in organic soluble form in a concentrated organic solvent solution;
• derivatizing said target analytes and analyte analogs present in said concentrated organic solvent solution to a derivatized form appropriate for gas chromatography/mass spectrometric analysis;
• performing a GC/MS analysis on said concentrated derivatized sample solution;
• and
• analyzing the results to quantitatively determine the amount of target analyte present in the meconium test specimen in terms of nanograms of THC-COOH analyte per gram of test specimen meconium by determining and multiplying the ratio of a response area of target analyte to a response area of the known analyte analog times the known quantity of the analyte analog added, divided by the mass of meconium test specimen.

A method for preparing a concentrated neonatal meconium extract containing substantially all of at least one target analyte present, if any, in a sample of infant meconium suspected of containing said at least one target analyte, said at least one target analyte is selected from the group consisting of amphetamine, cocaine metabolite, opiate and cannabinoids said concentrated meconium extract being specifically intended for use in subsequent qualitative and quantitative determinations of maternal use or exposure to said at least one target analyte during pregnancy and fetal exposure to said at least one target analyte in utero, said method comprising:

• providing a test sample of newborn meconium suspected of containing said at least one target analyte,
• adding a substantially non-aqueous, volatile organic acid to said sample to form a first mixture;
• agitating the first mixture for a time sufficient to extract substantially all of said at least one target analyte from the meconium sample, without the diluting the sample with water, until a substantially uniform first liquid homogenate is obtained;
• adding a volatile organic solvent to said first liquid homogenate to form a two-phase mixture including an organic phase and a second phase;
• thoroughly mixing the two-phase mixture for a time sufficient to permit substantially all of said extracted at least one target analyte to be extracted and collected into said organic phase;
• separating the organic phase from the second phase;
• and
• thereafter, reducing the volume of the separated organic phase to a fraction of its originally separated volume by evaporating at least a portion of said volatile organic solvent and acid components therefrom to provide said concentrated extract.