Chromatography–mass spectrometry. Cocaine metabolism in rats can be studied by injecting the drug and periodically with drawing blood to measure levels of metabolites by HPLC–mass spectrometry. For quantitative analysis, isotopically labelled internal standards are mixed with the blood sample. Blood was analysed by reversed-phase chromatography with an acidic eluent and atmospheric pressure chemical ionization mass spectrometry for detection. The mass spectrum of the collisionally activated dissociation products from the m/z 304 positive ion is shown in the figure on the next page. Selected reaction monitoring (m/z 304 from mass filter Q1 and m/z 182 from Q3 in Figure 22-33) gave a single chromatographic peak at 9.22 min for cocaine. The internal standard ${}^{{}^{\mathbf{2}}\mathbf{H}_{\mathbf{5}}}$-cocaine gave a single peak at 9.19 min for m/z 309 (Q1) 182(Q3).

(a) Draw the structure of the ion at m/z 304.

(b) Suggest a structure for the ion at m/z 182.

(c) The intense peaks at m/z 182 and 304 do not have ${}^{{}^{\mathbf{2}}\mathbf{C}}$isotopic partners at m/z 183 and 305. Explain why.

(d) Rat plasma is exceedingly complex. Why does the chromatogram show just one clean peak?

(e) Given that ${}^{{}^{\mathbf{2}}\mathbf{H}_{\mathbf{5}}}$-cocaine has only two major mass spectral peaks at m/z 309 and 182, which atoms are labelled with deuterium?

(f) Explain how you would use ${}^{{}^{\mathbf{2}}\mathbf{H}_{\mathbf{5}}}$-cocaine for measuring cocaine in blood.

Spectrum for Problem 25-25.

Left: Mass spectrum of collisionally activated dissociation products from m/z 304 positive ion from atmospheric pressure chemical ionization mass spectrum of cocaine.

Right: Chromatograms obtained by selected reaction monitoring. [Data from G. Singh, V. Arora, P. T. Fenn, B. Mets, and I. A. Blair, “Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry Assay for Trace Analysis of Cocaine and Its Metabolites in Plasma,” Anal. Chem. 1999, 71, 2021.]

Cocaine was analysed using the selected reaction monitoring method of chromatography-mass spectrometry. In this problem, the structure of fragments of the sample, and the structure of the ions that went through the first, second, and third quadrupoles were elucidated.

The reason why certain ions and certain isotopes were not detected, and how the chromatogram came to be were also studied. Also, the role of ${}^{{}^{\mathbf{2}}\mathbf{H}_{\mathbf{5}}}$-cocaine in the measurement of cocaine from a blood sample was also explored.

Part (a)

The given structure of cocaine in the problem has an m/z of 303, and is not charged. To get the structure of the positively charged ion with a m/z of 304 that was detected by the mass spectrometer, we simply bond a hydrogen atom to the nitrogen with a methyl group bonded to it:

Part (b)

A possible structure of the fragment with a m/z of 182 that the third quadrupole allows passage to the detector is:

After going through collisionally-activated dissociation, the ester substituent (-${}^{{\mathbf{COOC}}_{\mathbf{6}}{\mathbf{H}}_{\mathbf{5}}}$ -) and one hydrogen atom in cocaine could have been removed. Having a m/z of 122 altogether, the dissociation of this substituent and the hydrogen atom would leave the original structure with a m/z of 182.

Part (c)

The absence of M+1 peaks from the chromatogram is due to selected reaction monitoring. Because the first quadrupole and the third quadrupole only allowed structures with m/z 304 and m/z 182 respectively, the ions with these m/z were the only ones that reached the detector. The ions containing the ${}^{{}^{\mathbf{13}}\mathbf{C}}$isotopes, having a m/z of 305, were filtered out and not allowed access to the detector.

Part (d)

Like the ions with the ${}^{{}^{\mathbf{13}}\mathbf{C}}$isotopes, the ions that did not have a m/z of 304 or m/z of 182 were not able to reach the detector because they were filtered out by the quadrupoles used in the selected ion monitoring system.

Part (e)

Because, after fragmentation cocaine should still have a m/z of 182 despite having 5 deuterium ions (${}^{{}^{\mathbf{2}}\mathbf{H}}$ ),${}^{{}^{\mathbf{2}}\mathbf{H}_{\mathbf{5}}}$ should be bonded to the fragment that would be dissociated from cocaine to form an ion with an m/z of 182. Because the ester group fragment that would be dissociated from cocaine in (b) has 5 hydrogen atoms, the 5 deuterium ions should be located in this fragment so that when it gets dissociated, the remaining ion would have a m/z of 182.

Part (f)

To determine the concentration of cocaine in blood, what can be done is quantitative analysis based on the area of a chromatographic peak. Treating ${}^{{}^{\mathbf{2}}\mathbf{H}_{\mathbf{5}}}$-cocaine as an internal standard, the relationship of cocaine and ${}^{{}^{\mathbf{2}}\mathbf{H}_{\mathbf{5}}}$-cocaine is determined and quantified into the response factor. After determining the response factor using standard mixtures, the sample, which usually contains both the internal standard and the analyst, is fed into the chromatograph. Because the area of a peak is proportional to the quantity of the component it represents, equation 1 can be used to determine the amount of cocaine in a blood sample:

$\frac{{\mathbf{A}}_{\mathbf{x}}}{\left[X\right]}\mathbf{=}\mathbf{F}\frac{{\mathbf{A}}_{\mathbf{s}}}{\left[S\right]}$ (Equation 1)

where: ${}^{{\mathbf{A}}_{\mathbf{x}}}$is the area of the analyte signal,

${}^{\left[X\right]}$is the concentration of the analyte,

${}^{\mathbf{F}}$is the response factor,

${}^{{\mathbf{A}}_{\mathbf{s}}}$is the area of the internal standard peak,

${}^{\left[S\right]}$is the concentration of the standard.