OBJECTIVE

2

To establish a method for simultaneously determining three anti-tuberculosis drugs in the plasma of patients with spinal tuberculosis and apply it in clinical practice.

METHODS

2

LC-MS/MS method was established for the quantitative determination of the concentrations of isoniazid， rifampicin and pyrazinamide in the plasma of patients with spinal tuberculosis， using diphenhydramine as the internal standard. The determination was carried out using Chemalink CM-C

18

T column， with mobile phase consisting of 0.1% formic acid-methanol solution （gradient elution）， at the flow rate of 0.4 mL/min and an injection volume of 2 μL. Multiple reaction monitoring was conducted using an electrospray ionization source in positive ion mode. The ion pairs used for quantitative analysis were

m

/

z

138.0→121.0 （for isoniazid），

m

/

z

823.3→791.3 （for rifampicin），

m

/

z

124.1→79.0 （for pyrazinamide）， and

m

/

z

256.0→167.0 （for diphenhydramine）. Fifty-three patients diagnosed with spinal tuberculosis in Qinghai Provincial People’s Hospital from January 2023 to June 2025 were selected， and the plasma concentrations of isoniazid， rifampicin and pyrazinamide in these patients were measured using the above method.

RESULTS

2

The linear ranges for isoniazid， rifampicin and pyrazinamide were 0.5-16， 2-64

， and 2.5-80 μg/mL， respectively （

r

≥0.998 7）. The accuracy ranged from 90.20% to 108.64% （

n

＝5）. RSDs for intra-day precision were all less than 6.63% （

n

＝5）， while those for inter-day precision were all less than 8.42% （

n

＝3）. The matrix effects ranged from 88.60% to 115.41% （

n

＝5）. The relative deviations in the stability tests were all within the ±15% range， and the carry-over effect did not interfere with the determination. The results of clinical application showed that the mean plasma drug concentrations of isoniazid， rifampicin and pyrazinamide in patients with spinal tuberculosis were （3.62±2.80）， （8.55±4.57）， and （20.12±6.56） μg/mL， respectively. The incidences of plasma drug concentrations falling below the effective peak concentrations were 49.06%， 58.49% and 60.38%， respectively.

CONCLUSIONS

2

The method established in this study is rapid， accurate， and demonstrates good stability， making it suitable for clinical monitoring of the plasma concentrations of isoniazid， rifampicin and pyrazinamide in patients with spinal tuberculosis.