2.1. Development and Characterization of LC-ESI-MS Glycogen Analysis Methodology

Sodium hydroxide, 1-phenyl 3-methyl 5-pyrazolone (PMP), D-(+)-glucose-13C6 (≥99.0%), bovine liver glycogen, amyloglucosidase (Aspergillus niger), glacial acetic acid, and Tris hydrochloride were purchased from MilliporeSigma (Burlington, MA). Sodium acetate trihydrate, LC-MS grade methanol, and National C5000–1W 2mL clear glass ID Surestop vials was obtained from Thermo FisherScientific (Waltham, MA). Hipersolv chromanorm-acetonitrile was purchased from VWR Intl. (Atlanta, GA). Ammonium acetate was purchased from J.T. Baker-Avantor (Radnor, PA). High-purity formic acid and chloroform HPLC grade were obtained from VWR Intl. (Radnor, PA). Sodium bicarbonate was purchased from Spectrum Chemicals Mfg. Corp. (New Brunswick, NJ). Small volume (350μL) flat bottom borosilicate glass inserts (6 × 31 mm; AQ Brand) were obtained from Microsolv Technology Corp. (Leland, NC).

Accurately weighed 10.0 mg D-(+)-Glucose was dissolved in 1.0 mL water; after vigorous shaking, a 100 μL aliquot was added to 0.5 M methanolic PMP (100 μL) and 0.3 M sodium hydroxide (100 μL), then heated to 70° C (30 min.), followed by cooling to room temperature [23, 24]. After centrifugation at 5000 rpm (2 min.), excess sodium hydroxide was neutralized with 400 μL 0.2 M formic acid. Samples were shaken, extracted with 500 μL chloroform, vacuum-concentrated (30 min; 45° C) for removal of chloroform and formic acid, transferred to fresh tubes, frozen at −80°C (30 min.), and lyophilized. The lyophilization product was diluted to 1.0 mL with 10 mM ammonium acetate, bath-sonicated (30 sec.), and centrifuged. Supernatant aliquots (250 μL) were transferred to 350 μL inserts, which were placed into 2 mL Surestop vials in an autosampler tray. D-(+)-Glucose-PMP derivative was measured by LC-MS at m/z 510.2. The reaction scheme is presented in Figure 1. A calibration curve ranging from 62.5 to 1000 μg D-(+)-Glucose-PMP/mL was developed.

Glucose reacts with 1-phenyl-3-methyl-5-pyrazolone in mild alkaline conditions, forms glucose-PMP derivative. This derivative can be detected in negative mode at m/z 510.2.

After preparation of a stock solution of glycogen in water (2 mg/mL), diluted stock aliquots (20 μL) containing 0.625 or 40.0 μg glycogen were transferred to tubes containing 0.1M sodium acetate, pH 5.0 (10μL) and 0.5 mg/ml amyloglucosidase (10μL) [Fuller et al]. Contents were vortexed (30 sec.), centrifuged, and incubated at 37° C (2 hr). Hydrolysis reactions were terminated by heating to 100° C (5 min). Tubes were cooled to room temperature, centrifuged at 5000 rpm (2 min), wrapped with perforated aluminum foil, frozen at −80° C (30 min), and lyophilized overnight at −55° C. Lyophilized samples were treated with 100μL 0.5M methanolic PMP and 100μL 0.3M sodium hydroxide, then processed according to sequential steps described above. PMP derivative of released glucose was diluted to 1 mL with 10 mM ammonium acetate.

An assembled chromatography system [UHPLC Vanquish binary pump (prod. no. VFP10A01/121345), Vanquish auto-sampler (prod. no. VFA10A02/121345), and temperature-controlled Vanquish UHPLC+ column compartment (prod. no. VHC10A02/121345)] was coupled to an ISQ EC mass spectrometer (prod. no. ISQECLC/121345) (ThermoFisherScientific, Waltham, MA). Two chromatographic [Acclaim TM 120 C18 (4.6 mm ID × 100 mm L, 5μm, 120Å; prod. no. 059147, ThermoFisherSci.) and Shodex™ Asahipak™ NH2P-4 3E (3.0 mm ID × 250 mm L); no. M17T0005, Midland Scientific, Inc., La Vista, NE] columns were used to optimize mass spectrometric parameters for detection of D-(+)-Glucose-PMP. Column and autosampler temperatures were 35° C and 15° C, respectively. The auto-sampler needle was washed with 10% (v/v) methanol (10 sec). ThermoScientific™ Dionex™ Chromeleon™ 7 Chromatography Data System software (prod. no. 7200.0300/121345) was used for mass spectrometric analysis.

Analysis of D-(+)-Glucose-PMP was performed in negative ionization mode. D-(+)-Glucose-PMP ion chromatograms were extracted from Total Ion Current (TIC) at m/z 510.2 to generate area-under-the-curve data. Parameters for sheath gas pressure (SGP; 25 psig), auxiliary gas pressure (AGP; 4.6 psig), sweep gas pressure (SWGP; 0.5 psig), vaporizer temperature (VT; 150° C), ion transfer tube temperature (ITT; 150° C), source voltage (−2000V), foreline pressure (1.76 Torr; auto-set by instrument- and variable), source gas (nitrogen; Genius NM32LA 110V, 10–6520; Peak Scientific, Inchinnan, Scotland), and mass peak area detection algorithm (ICIS/Genesis) were maintained at optimum. The mass scan range was between 170 and 700.

Acetonitrile: 10 mM ammonium acetate (25:75, v/v) was run as mobile phase, at a 0.5 mL/min flow rate, over a 20 min. run interval. Column compartment and auto-sampler temperatures were set at 40° C and 15° C, espectively. Mass spectrometry source settings were set as follows: VT=282° C, ITT=300° C, SGP=49.9psig, AGP=5.7 psig, SWGP=0.5psig. M/z scans, e.g. 173–176, 505–515, in negative mode and positive mode were run with source voltage −2000V and 3000V, respectively. Chromatograms showing intended peaks occurred only in negative mode. Post-injection recording of 10 μL 12.48 ug D-(+)-Glucose-PMP/mL acetonitrile:10 mM ammonium acetate (25:75, v/v) was carried out for 20 min [Figure 1, Panels A–C]. Shodex™ Asahipak™ NH2P-40 3E column: Acetonitrile:10 mM ammonium acetate (75:25, v/v) was run as mobile phase at column compartment and auto-sampler temperatures of 35° C and 15° C, respectively. Source settings were as follows: VT=70° C, ITT=200° C, SGP=25psig, AGP=5psig, SWGP=0.5psig, and source voltage −2000V and 3000V, respectively. Post-injection recording of 10μL of 50μg D-(+)-Glucose-PMP/mL was performed over a 40 min interval; results were obtained only in negative scan mode [Figure 2, Panels D and ​andE].E]. After attaining a two-fold improvement of D-(+)-Glucose-PMP detection using the Shodex™ Asahipak™ NH2P-40 3E column, various gas and temperature parameters were manipulated in an effort to further enhance detection [Figure 2], while sample injection volume, flow rate and mobile phase composition were kept constant. TIC and extracted chromatograms obtained at m/z 510.2 are depicted in Figures 3 and ​and4,4, respectively. Preliminary optimization revealed that VT=150° C, ITT=150° C, SGP=25psig, AGP=4.6psig, SWGP=0.5psig produced greatest peak area for D-(+)-Glucose-PMP at 510.2.

A) Representative total ion current (TIC) chromatogram of D-(+)-Glucose-PMP 12.48μg/mL in C18 column, Inj. Volume: 10μL, showing very low chromatographic sensitivity at retention time 2.1 min compared to PMP alone (large PMP peak shown in box at upper right-hand corner) at 10 min. B) Extracted D-(+)-Glucose-PMP showed very low chromatographic area at 2.1 min and poor resolution. C) Extracted PMP has very high chromatographic area at retention time 10 min and can be detected easily over D-(+)-Glucose-PMP in C18 column, indicating unsuitability of this column for identification of D-(+)-Glucose-PMP. D) TIC chromatogram of D-(+)-Glucose-PMP 50μg/mL in NH2P-40 3E column, Inj. Vol: 10μL, shows D-(+)-Glucose-PMP detection with distinguishing chromatographic peak at retention time 3 min in NH2P-40 3 E column. E) Extracted chromatogram of D-(+)-Glucose-PMP in NH2P-40 3E column at m/z 510.2 shows sele.ctive D-(+)-Glucose-PMP at retention time 3.5 min.

Effects of vaporizer temperature (VT), ion transfer tube temperature (ITT), sheath gas pressure (SGP), aux gas pressure (AGP), and sweep gas pressure (SWGP), described in Methods 1–9, are shown in corresponding TIC D-(+)-Glucose-PMP chromatograms 1–9.

Extracted chromatograms 1–9 of D-(+)-Glucose-PMP 50μg/mL, Inj.Vol: 10μL. Mass spectrometric parameters of Method 9 show highest D-(+)-Glucose-PMP peak area in NH2P-40 3E column using Method 9.

Various combinations of the AGP, SGP, ITT, and VT at constant SWGP and ITT were evaluated to determine maximal area at constant SWGP=0.5 psig and ITT=150°C. The response parameter was peak area with respect to a linear increase in VT at various levels of AGP and SGP [Figure 5].

A) At AGP 2, SGP 50 psig yielded highest peak area at vaporizing temperature (VT) 200°C compared to 14.7, 25, or 75 SGP, respectively. SGP 50 psig caused a poor response at VT 50°C. Most chromatograms showed split peaks. B) At AGP 5, a distinct chromatographic peak without splits were observed at SGP 25 psig, VT 150°C, though peak area was medium when compared to SGP 75 psig and 14.7 psig. C) At AGP 10, SGP 50 and 75 psig, peak area degrades at ITT and 150°C VT; most data had split peaks and were not selected for further analyses.

Reduction of sample injection volume from 10μL to 2μL resulted in a clean TIC chromatogram without background. Mass method parameters were set at AGP=4.6 psig, SGP=25 psig, SWGP=0.5 psig, VT=150°C, and ITT=150°C, respectively. D-(+)-Glucose-PMP (2μL; 1000μg/mL or 62.5μg/mL) and hydrolyzed glycogen diluted with 10mM ammonium acetate (2μL; 40μg/mL or 0.625μg/mL) were injected separately. A flow rate of 0.25mL/min, mobile phase 75:25v/v, acetonitrile: 10mM ammonium acetate, and Shodex™ Asahipak™ NH2P-40 3E column were used. Column and autosampler temperatures were 35° C and 15° C, respectively. TIC and extracted chromatograms are shown in Figures 6 and ​and7,7, respectively.

AGP 4.6 psig, SGP 25 psig, SWGP 0.5 psig, VT 150°C, and ITT 150°C were selected for the analysis of D-(+)-Glucose-PMP. Representative chromatograms here show lack of D-(+)-Glucose-PMP background from standard (at left) or hydrolyzed glycogen (at right) when inj. vol. less than or equal to 2μL.

Extracted chromatograms show peaks with highest and least D-(+)-Glucose-PMP area from standard (at left) and hydrolyzed glycogen (at right) with retention time 5 min. Different concentrations were selected to generate a linear equation for glucose and glycogen.

The D-(+)-Glucose-13C6 failed to yield a reproducible response, and degraded after 7hr of residence in autosampler at 15°C, and was measured at [M-H] m/z 515.2 [Figure 8]. A high IS concentration, e.g. 4mg/mL, was required to elicit a minimal response at AGP=4.6 psig, SGP=25 psig, SWGP=0.5 psig, VT=150°C, and ITT=150°C.

Mass spectrometric parameters of AGP 4.6 psig, SGP 25 psig, SWGP 0.5 psig, VT 150°C, and ITT 150°C selected for the analysis of internal standard (IS) 13C6-D-(+)-Glucose-PMP. A high IS concentration of 4mg/mL was selected, but failed to show a good response at 0 h and was destabilized after 7 h.