Gas chromatography–electron capture negative chemical ionization–MS (reasoning & description)

Function of a GC-MS = Identify the different components within a test sample.

Objective: To find out the percentage enrichment of ²H-labelled retinol in circulation by measuring retinol enrichment from the reference dose and yellow maize .

The total enrichment of labeled retinol was determined by evaluation of the negative ions.

GLC is connected to MS to enhance its capability for qualitative analysis.

Sample molecules are ionized at the ionization source where the chemical bonds are broken down to form fragments.

The ions are sorted and detected. A mass spectrum with ions of different mass-to-charge ratio is plotted.

400 μL of the serum sample was extracted (as described in the HPLC analysis of serum samples) and injected into HPLC equipped with a C₁₈ column [Reversed Phase].

Retinol retrieved from the HPLC system was dried in nitrogen gas, the residue was chemically derivatized to form retinyl trimethylsilyl ether.

2–3 μL was injected into the gas chromatography (GC)–MS system after cooling.

GC-MS instrument : Agilent 6890

Type of Detector: 5973 Network Mass Selective Detector (linear dynamic range of 10⁴)

Type of GC column: Zebron ZB-1MS capillary

Carrier Gas: Helium

Reaction Gas: Methane

GC oven temperature: Increased from 50°C to 220°C at a speed of 15°C/min, to 230°C at 5°C/min, and to 310°C at 20°C/min; then it remained at 310°C for 5 min before it was cooled down to 50°C . (Programme temperature-stepwise mode)

-> trans retinol peak with the retention time of 12 min is produced.

Mass spectrometer was set at a mass-to-charge ratio (m/z) of 260–280.


The m/z of M_retinol +4 and M_retinol +5 = m/z 272 and 273 could be distinctly detected and combined to represent the formation of retinol from the labeled maize β-carotene.

Enrichment of ²H-labeled retinol from yellow maize β-carotene:

The enrichment of ¹³C retinol from [¹³C₁₀] retinyl acetate (reference dose) was also calculated in the same fashion.

LC-atmospheric pressure chemical ionization-MS 

Objective: To find out the percentage enrichment of labeled β-carotene in yellow maize and in human serum
 

{Molecular mass of unlabeled β-carotene in positive-mode LC-MS analysis: 

537 (M+H⁺), 538 (M+H⁺, ¹³C β-C), and 539 (M+H⁺, ¹³C₂ β-C) }

To ensure maximal detection of labeled β-carotene in serum, only the molecular mass of labeled β-carotene isotopomers were analysed: 

544 (M+ H⁺ +7), 545 (M+ H⁺+8), 546 (M+ H⁺+9), 547 (M+ H⁺+10), and 548 (M+ H⁺+11)

Determining the relative abundance of m/z 544–548....
There are 60% of β-carotene that are labelled in the entire yellow maize.

Further calculations...

[Total serum volume (estimated at 0.0435 L per kilogram body weight)]

x

[Concentration of [²H]β-carotene and [²H]retinol and [¹³C₁₀] retinol in the circulation (nmol/L)]

= Total serum responses (nmol) to the [²H]β-carotene dose and the [¹³C₁₀]retinyl acetate dose

Curves of total serum responses (expressed as nmol on the y axis) plotted against time (expressed as days on the x axis)

Areas under the curve (AUC) in nmol · d are determined for

->the serum labeled retinol responses after the ingestion of [²H] β-carotene dose

->  the [¹³C₁₀]retinyl acetate dose

M_retinol = 291 for [²H]retinol while M_retinol = 296 for [¹³C₁₀]retinol

AUCs were calculated for 21 d after each labeled tracer due to the 7-d delay in the administration of the retinyl acetate dose.

Retinol equivalence calculations

Compare

->->AUC of serum [²H]retinol response from labeled yellow maize β-carotene

->->AUC of 1 mg [¹³C₁₀]serum response

[²H] retinol from [²H] yellow maize β-carotene (nmol):