Determination of glucose in human aqueous humor using Raman spectroscopy and designed-solution calibration

Glucose concentrations of in vitro human aqueous humor (HAH) samples from cataract patients were determined using 785 nm Raman spectra and partial least squares (PLS) calibration. PLS models were created from spectra of prepared calibration solutions rather than aqueous humor samples. Spectra were obtained with an excitation energy (100 mW for 150 s), which was higher than can be applied in vivo, to decrease the models' contribution to prediction uncertainty. The solutions contained experimentally designed levels of glucose, bicarbonate, lactate, urea, and ascorbate. Multiplicative signal correction of spectra helped compensate for the +/-20% drift in laser power observed at the sample over six noncontiguous days of data collection. Seventeen HAH samples containing 38-775 mg/dL of glucose exhibited a root-mean-square error (RMSEP) of 22 mg/dL, coefficient of determination (r(2)) of 0.989, and bias of 6 mg/dL when predicted from lower energy (30 s) spectra collected contemporaneously with fifty calibration spectra. Similar results were obtained even when spectral data were gathered separately for human aqueous humor samples and calibration samples: 10 HAH samples, calibrated on 25 solutions measured 3.6 weeks earlier, exhibited an RMSEP of 23 mg/dL, r(2) of 0.992, and bias of 9 mg/dL. The results demonstrate progress toward the determination of glucose levels in patient-derived aqueous humor using laboratory-derived "artificial aqueous humor" calibration solutions.