Question

In 2002, workers at the Swedish National Food Administration discovered that heated, carbohydrate-rich foods, such as french fries, potato chips, and bread, contain alarming levels \((0.1to4\mu {\rm{g}}/{\rm{g}})\) of acrylamide, a known carcinogen\(36\).

After the discovery, simplified methods were developed to measure ppm levels of acrylamide in food. In one procedure,\(10\;{\rm{g}}\) of pulverized, frozen french fries were mixed for \(20\;{\rm{min}}\)with\(50\;{\rm{mL}}\) of \({{\rm{H}}_2}{\rm{O}}\)to extract acrylamide, which is very soluble in water \((216\;{\rm{g}}/100\;{\rm{mL}}).\)The liquid was decanted and centrifuged to remove suspended matter. The internal standard \(^2{{\rm{H}}_3}\)-acrylamide was added to\(1\;{\rm{mL}}\) of extract. A solid-phase extraction column containing \(100{\rm{mg}}\)of cation-exchange polymer with protonated sulfonic acid groups\(\left( { - {\rm{S}}{{\rm{O}}_3}{\rm{H}}} \right.)\) was washed twice with 1 -mL portions of methanol and twice with \(1 - {\rm{mL}}\)portions of water. The aqueous food extract \((1{\rm{mL}})\)was then passed through the column to bind protonated acrylamide \(\left( { - {\rm{NH}}_3^ + } \right)\)to sulfonate \(\left( { - {\rm{SO}}_3^ - } \right)on\)the column. The column was dried for\(30\;{\rm{s }}at\)\(0.3\)bar and then acrylamide was eluted with\(1\;{\rm{mL}}\) of \({{\rm{H}}_2}{\rm{O}}.\)Eluate was analyzed by liquid chromatography with a polar bonded phase. The chromatograms show the results moni- tored by ultraviolet absorbance or by mass spectrometry. The retention time of acrylamide is different on the two columns because they have different dimensions and different flow rates.

(a) What is the purpose of solid-phase extraction prior to chromatography? How does the ion-exchange sorbent retain acrylamide?

(b) Why are there many peaks when chromatography is monitored by ultraviolet absorbance?

(c) Mass spectral detection used selected reaction monitoring (Figure 22-33) with the \(m/z72 \to 55\)transition for acrylamide and \(75 \to 58fo{r^2}{{\rm{H}}_3}\)-acrylamide. Explain how this detection method works and suggest structures for the ions with \({\rm{m}}/{\rm{z}}72\)and 55 from acrylamide.

(d) Why does mass spectral detection give just one major peak?

(e) How is the internal standard used for quantitation with mass spectral detection?

(f) Where does \(^2{{\rm{H}}_3}\)-acrylamide appear with ultraviolet absorbance? With mass spectral selected reaction monitoring?

(g) Why does the mass spectral method give quantitative results even though retention of acrylamide by the ion-exchange sorbent is not quantitative and elution of acrylamide from the sorbent by \(1\;{\rm{mL}}\) of water might not be quantitative?

Chromatograms of acrylamide extract after passage through solid-phase extraction column. Left: Phenomenex Synergi Polar-RP 4- \(\mu {\rm{m}}\)column eluted with 96:4 \((vol/vol){{\rm{H}}_2}{\rm{O}}:{\rm{C}}{{\rm{H}}_3}{\rm{CN}}.\)Right: Phenomenex Synergi Hydro-RP 4- \(\mu {\rm{m}}\)column eluted with \(96:4:0.1(vol/vol/vol){{\rm{H}}_2}{\rm{O}}:{\rm{C}}{{\rm{H}}_3}{\rm{OH}}:{\rm{HC}}{{\rm{O}}_2}{\rm{H}}.\) (Data from L. Peng. T. Farkas, L. Loo, \({\rm{J}}.\)Teuscher, and \({\rm{K}}.\)Kallury, "Rapid and Reproducible Extraction of Acrylamide in French Fries Using a Single Solid-Phase Sorbent," Am. Lab. News Ed, October 2003, p. 10.)

Short answer

a. Solid phase extraction involves separation of mixture into individual compounds.

b. Many components absorb ultraviolet light and creates a large number of peaks.

c. Structure for the given ions are

\({\rm{C}}{{\rm{H}}_2} = {\rm{CHCON}}{{\rm{H}}_3}^ + ({\rm{m}}/{\rm{z}} = 72) \to {\rm{C}}{{\rm{H}}_2} = {\rm{CHC}} \equiv {{\rm{O}}^ + }({\rm{m}}/{\rm{z}} = 55)\)

\({\rm{C}}{{\rm{D}}_2} = {\rm{CDCON}}{{\rm{H}}_3}^ + ({\rm{m}}/{\rm{z}} = 75) \to {\rm{C}}{{\rm{D}}_2} = {\rm{CDC}} \equiv {{\rm{O}}^ + }({\rm{m}}/{\rm{z}} = 58)\)

d. The sole \({\rm{m}}/{\rm{z}}72\)is acrylamide that produces \({\rm{m/z 55 }}{\rm{.}}\)

e. \((acrylamide))(internalstandard) = (areaof{\rm{m}}/{\rm{z}}72 \to 55)/(areaof{\rm{m}}/{\rm{z}}75 \to 58).\)

f. \(^2{{\rm{H}}_3}\) -acrylamide appears same time as UV absorbance.

g. Both the fractions are equal.

Step-by-step solution

Concept used

Solid-phase extraction is a sample preparation technique. The physical and chemical characteristics of the compounds that are dissolved or suspended in a liquid mixture and separated are determined by their physical and chemical properties.

The purpose of solid-phase extraction

(a)

Solid-phase extraction is nothing more than sample preparation that can lead to the separation of a mixture of chemicals into individual compounds.

While passing through several additional components in the solid-phase extraction, \(^{{\rm{C}}{{\rm{H}}_2} = {\rm{CHCON}}{{\rm{H}}_3} + }\)can be retained.

Monitored by ultraviolet absorbance

(b)

- Normally, UV absorbance is used to check chromatography. The components of a chromatography sample can easily absorb ultraviolet radiation.

- As a result, numerous components absorb ultraviolet light and produce a large number of peaks in chromatography when analysed using ultraviolet absorhance.

how this detection method works:

(c)

The following is how the process works.

The mass spectrum for acrylamide is shown in the transition.

\({\rm{m}}/{\rm{z}}72to55.\)

To establish the structures for \({\rm{m}}/{\rm{z}}72\)and 75, we must first identify the transition \({\rm{m}}/{\rm{z}}72to55\)

The \({\rm{m}}/{\rm{z}}72,\)which is chosen by \({\rm{Q}}1\) and can dissociate the sample by collisions in \({\rm{Q}}2.\)The product for\({\rm{m}}/{\rm{z}}55\) can be selected in \({\rm{Q}}3\)for detection purposes.

The following are some possible structures.

\({\rm{C}}{{\rm{H}}_2} = {\rm{CHCON}}{{\rm{H}}_3}^ + ({\rm{m}}/{\rm{z}} = 72) \to {\rm{C}}{{\rm{H}}_2} = {\rm{CHC}} \equiv {{\rm{O}}^ + }({\rm{m}}/{\rm{z}} = 55)\)

\({\rm{C}}{{\rm{D}}_2} = {\rm{CDCON}}{{\rm{H}}_3}^ + ({\rm{m}}/{\rm{z}} = 75) \to {\rm{C}}{{\rm{D}}_2} = {\rm{CDC}} \equiv {{\rm{O}}^ + }({\rm{m}}/{\rm{z}} = 58)\)

mass spectral detection:

(d)

Because acrylamide is the only \({\rm{m}}/{\rm{z}}72\) which can produces \({\rm{m/z 55 }}{\rm{.}}\)

internal standard used for quantitation:

(e)

The internal standard that may be utilised for quantification in mass spectral detection is expressed as follows.

\((acrylamide))(internalstandard) = (area of {\rm{m}}/{\rm{z}}72 \to 55)/(areaof{\rm{m}}/{\rm{z}}75 \to 58).\)

-acrylamide appear with ultraviolet absorbance:

(f)

Ultraviolet absorbance: - Whatever signals are released in the ultraviolet absorption happen all at once It looks to be\(^2{{\rm{H}}_3}\) -acrylamide.

SRM: Neither the acrylamide nor the internal standard are stopped by the detector in this approach, therefore there is no chance of interference.

Why does the mass spectral method give quantitative results

(g)

- The recovered quantities of acrylamide fraction and internal standard fraction are same. The fraction produced by acrylamide and the fraction obtained by internal standard, both of which are bound and recovered from a solid-phase extraction column, are almost identical.