New HPLC method for determination of cystatin C biomarker in human blood

Abstract

A rapid and sensitive method for determination of cystatin C (CC) protein in human blood by means of high performance liquid chromatography (HPLC) was developed and validated. Estimated glomerular filtration rate (eGFR) has been calculated for the patients with chronic kidney disease (CKD). Ion pair liquid chromatography technique was utilized to separate CC along with UV detection. Calibration curve with excellent correlation (r 2 = 0.99) over the range from 0.75 to 20.50 mg/L of CC was accomplished. Limits of detection and quantifycation were 0.375 and 0.75 mg/L of CC, respectively. The recoveries were in the range of 93.6-102.3%. Intra-assay and inter-assay variabilities were 8.2 and 6.14%, respectively.

Influence of age, gender, smoking, diabetes, thyroid and cardiac dysfunctions on cystatin C biomarker

“…From the statistical data, a significant relationship has been confirmed for the increased levels of CC in the hyperthyroidism subjects (p = 0.001) (Figure 1). On the other hand, a decrease in CC levels was observed in hypothyroidism cases (0.40 to 1.42 with an average of 0.91, normal 0.47 to 1.87) mg/L, furthermore, CC levels in 3 samples were even below the detection limit of the method (entries, 2-4, supplemental table 9) 31 .…”

“…Centrifugation was done at the same parameters for 30 minutes and the supernatant was filtered through 0.45 µm teflon filter prior to injection into the HPLC system 31 .…”

“…The injection volume was 100 µL. The eluent was monitored at 224 nm (for detailed validation tests refer to our previous published work 31 ).…”

“…Due to several disadvantages of the available immunoassay methods; Particle enhanced nephelometric immunoassay (PENIA) 6 , particle enhanced turbidimetric method (PET) 29 and enzyme-amplified single radial immunodiffusion 30 , a new high performance liquid chromatographic (HPLC) method has been developed by our group and used for the quantification purpose of CC 31 . It should be noted, that there are two HPLC-Mass analytical methods for the determination of CC, however, one of them was only used for raw material 32 and in the another one no real biological samples were introduced 33 .…”

Influence of age, gender, smoking, diabetes, thyroid and cardiac dysfunctions on cystatin C biomarker

“…From the statistical data, a significant relationship has been confirmed for the increased levels of CC in the hyperthyroidism subjects (p = 0.001) (Figure 1). On the other hand, a decrease in CC levels was observed in hypothyroidism cases (0.40 to 1.42 with an average of 0.91, normal 0.47 to 1.87) mg/L, furthermore, CC levels in 3 samples were even below the detection limit of the method (entries, 2-4, supplemental table 9) 31 .…”

“…Centrifugation was done at the same parameters for 30 minutes and the supernatant was filtered through 0.45 µm teflon filter prior to injection into the HPLC system 31 .…”

“…The injection volume was 100 µL. The eluent was monitored at 224 nm (for detailed validation tests refer to our previous published work 31 ).…”

“…Due to several disadvantages of the available immunoassay methods; Particle enhanced nephelometric immunoassay (PENIA) 6 , particle enhanced turbidimetric method (PET) 29 and enzyme-amplified single radial immunodiffusion 30 , a new high performance liquid chromatographic (HPLC) method has been developed by our group and used for the quantification purpose of CC 31 . It should be noted, that there are two HPLC-Mass analytical methods for the determination of CC, however, one of them was only used for raw material 32 and in the another one no real biological samples were introduced 33 .…”

Influence of age, gender, smoking, diabetes, thyroid and cardiac dysfunctions on cystatin C biomarker

“…From the statistical data, a significant relationship has been confirmed for the increased levels of CC in the hyperthyroidism subjects (p = 0.001) (Figure 1). On the other hand, a decrease in CC levels was observed in hypothyroidism cases (0.40 to 1.42 with an average of 0.91, normal 0.47 to 1.87) mg/L, furthermore, CC levels in 3 samples were even below the detection limit of the method (entries, 2-4, supplemental table 9) 31 .…”

“…Centrifugation was done at the same parameters for 30 minutes and the supernatant was filtered through 0.45 µm teflon filter prior to injection into the HPLC system 31 .…”

“…The injection volume was 100 µL. The eluent was monitored at 224 nm (for detailed validation tests refer to our previous published work 31 ).…”

“…Due to several disadvantages of the available immunoassay methods; Particle enhanced nephelometric immunoassay (PENIA) 6 , particle enhanced turbidimetric method (PET) 29 and enzyme-amplified single radial immunodiffusion 30 , a new high performance liquid chromatographic (HPLC) method has been developed by our group and used for the quantification purpose of CC 31 . It should be noted, that there are two HPLC-Mass analytical methods for the determination of CC, however, one of them was only used for raw material 32 and in the another one no real biological samples were introduced 33 .…”

Influence of age, gender, smoking, diabetes, thyroid and cardiac dysfunctions on cystatin C biomarker

“…From the statistical data, a significant relationship has been confirmed for the increased levels of CC in the hyperthyroidism subjects (p = 0.001) (Figure 1). On the other hand, a decrease in CC levels was observed in hypothyroidism cases (0.40 to 1.42 with an average of 0.91, normal 0.47 to 1.87) mg/L, furthermore, CC levels in 3 samples were even below the detection limit of the method (entries, 2-4, supplemental table 9) 31 .…”

“…Centrifugation was done at the same parameters for 30 minutes and the supernatant was filtered through 0.45 µm teflon filter prior to injection into the HPLC system 31 .…”

“…The injection volume was 100 µL. The eluent was monitored at 224 nm (for detailed validation tests refer to our previous published work 31 ).…”

“…Due to several disadvantages of the available immunoassay methods; Particle enhanced nephelometric immunoassay (PENIA) 6 , particle enhanced turbidimetric method (PET) 29 and enzyme-amplified single radial immunodiffusion 30 , a new high performance liquid chromatographic (HPLC) method has been developed by our group and used for the quantification purpose of CC 31 . It should be noted, that there are two HPLC-Mass analytical methods for the determination of CC, however, one of them was only used for raw material 32 and in the another one no real biological samples were introduced 33 .…”