Establishment and validation of the LC-MS/MS method for the determination of lincomycin in human blood: Application to an allergy case in forensic science.

An analytical method to quantify lincomycin in human blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. The selected method was based on a protein precipitation extraction (PPE) with methanol. Instrumental determination was carried out by LC-MS/MS, with quantification based on the internal standard method. Linearity for lincomycin was established in the concentration range of 5-100 ng/mL. The limit of detection (LOD) and limit of quantification (LOQ) were 0.2 and 1 ng/mL, respectively. Analyte recoveries were in the range of 72.70%-84.13% for spiked blood samples. The accuracies ranged between 92.82% and 100.40%, and the intraday and inter-day precisions ranged between 1.19% and 6.40%, respectively. The developed method was applied to an authentic allergy case of lincomycin. By testing the lincomycin content in the venous blood of the deceased and combined with the pathological test results, lincomycin acute allergy appeared to be the most likely cause of death. The acquired results confirm that the developed method is capable of identifying and quantifying lincomycin in human blood and can be suitable for the detection of allergy cases in clinical or forensic science.

Development and validation of LC-MS/MS methods to measure tobramycin and lincomycin in plasma, microdialysis fluid and urine: application to a pilot pharmacokinetic research study.

Background The aim of our work was to develop and validate a hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) methods for the quantification of tobramycin (TMC) and lincomycin (LMC)in plasma, microdialysis fluid and urine. Methods Protein precipitation was used to extract TMC and LMC from plasma, while microdialysis fluid and urine sample were diluted prior to instrumental analysis. Mobile phase A consisted of 2 mM ammonium acetate in 10% acetonitrile with 0.2% formic acid (v/v) and mobile phase B consisted of 2 mM ammonium acetate in 90% acetonitrile with 0.2% formic acid (v/v). Gradient separation (80%-10% of mobile phase B) for TMC was done using a SeQuant zic-HILIC analytical guard column. While separation of LMC was performed using gradient elution (100%-40% of mobile phase B) on a SeQuant zic-HILIC analytical column equipped with a SeQuant zic-HILIC guard column. Vancomycin (VCM) was used as an internal standard. A quadratic calibration was obtained over the concentration range for plasma of 0.1-20 mg/L for TMC and 0.05-20 mg/L for LMC, for microdialysis fluid of 0.1-20 mg/L for both TMC and LMC, and 1-100 mg/L for urine for both TMC and LMC. Results For TMS and LMC, validation testing for matrix effects, precision and accuracy, specificity and stability were all within acceptance criteria of ±15%. Conclusions The methods described here meet validation acceptance criteria and were suitable for application in a pilot pharmacokinetic research study performed in a sheep model.

High-Precision Quantitation of Biofluid Samples Using Direct Mass Spectrometry Analysis.

The transition of mass spectrometry for clinical analysis is highly desirable, and major progress has been made with direct sampling ionization for operation simplification. High-precision quantitation, however, remains a major challenge in this transition. Herein, a novel method was developed for direct quantitation of biofluid samples, using an extremely simplified procedure for incorporation of internal standards selected against the traditional rules. Slug flow microextraction was used for the development, with conditions predicted by a theoretical model, viz., using internal standards of partition coefficients very different from the analytes and large sample-to-extraction solvent volume ratios. Direct quantitation of drug compounds in urine and blood samples was demonstrated. This development enabled an extremely simplified protocol that is expected to have a significant impact on on-site or clinical analysis.

Pharmacokinetics of lincomycin following single intravenous administration in buffalo calves.

Lincomycin 10 mg kg(-1), IV in buffalo calves followed two-compartment open model with high distribution rate constant α (11.2 ± 0.42 h(-1)) and K 12/K 21 ratio (4.40 ± 0.10). Distribution half-life was 0.06 ± 0.01 h and AUC was 41.6 ± 1.73 µg mL(-1) h. Large Vdarea (1.15 ± 0.03 L kg(-1)) indicated good distribution of lincomycin in various body fluids and tissues. Peak plasma level of lincomycin (71.8 ± 1.83 µg mL(-1)) was observed at 1 min as expected by IV route. The elimination half-life and MRT of lincomycin were short (3.30 ± 0.08 and 4.32 ± 0.11 h, respectively). Lincomycin 10 mg kg(-1) IV at 12-h interval would be sufficient to maintain T > MIC above 60 % for bacteria with minimum inhibitory concentrations (MIC) values ≤1.6 µg mL(-1). Favourable pharmacokinetic profile in buffalo calves and a convenient dosing interval suggest that lincomycin may be an appropriate antibacterial in buffalo species for gram-positive and anaerobic bacterial pathogens susceptible to lincomycin.

Pharmacokinetics and bone tissue concentrations of lincomycin following intravenous and intramuscular administrations to cats.

The pharmacokinetic properties and bone concentrations of lincomycin in cats after single intravenous and intramuscular administrations at a dosage rate of 10 mg/kg were investigated. Lincomycin minimum inhibitory concentration (MIC) for some gram-positive strains isolated from clinical cases was determined. Serum lincomycin disposition was best-fitted to a bicompartmental and a monocompartmental open models with first-order elimination after intravenous and intramuscular dosing, respectively. After intravenous administration, distribution was rapid (T(1/2(d)) = 0.22 ± 0.09 h) and wide as reflected by the volume of distribution (V((d(ss)))) of 1.24 ± 0.08 L/kg. Plasma clearance was 0.28 ± 0.09 L/h · kg and elimination half-life (T(1/2)) 3.56 ± 0.62 h. Peak serum concentration (C(max)), T(max), and bioavailability for the intramuscular administration were 7.97 ± 2.31 µg/mL, 0.12 ± 0.05 h, and 82.55 ± 23.64%, respectively. Thirty to 45 min after intravenous administration, lincomycin bone concentrations were 9.31 ± 1.75 µg/mL. At the same time after intramuscular administration, bone concentrations were 3.53 ± 0.28 µg/mL. The corresponding bone/serum ratios were 0.77 ± 0.04 (intravenous) and 0.69 ± 0.18 (intramuscular). Lincomycin MIC for Staphylococcus spp. ranged from 0.25 to 16 µg/mL and for Streptococcus spp. from 0.25 to 8 µg/mL.

A flow injection chemiluminescence method for the determination of lincomycin in serum using a diperiodato-cuprate (III)-luminol system.

In this paper, the novel trivalent copper-periodate complex {K5[Cu(HIO6)2], DPC} has been applied in a luminol-based chemiluminescence (CL) reaction. Coupled with flow injection (FI) technology, the FI-CL method was proposed for the determination of lincomycin hydrochloride. The CL reaction between luminol and DPC occurred in an alkaline medium. The CL intensity could be greatly enhanced by lincomycin hydrochloride. The relative CL intensity was proportional to the concentration of lincomycin hydrochloride in the range of 1 × 10⁻8 to 5 × 10⁻6 g mL⁻¹ and the detection limit was at the 3.5 × 10⁻9 g mL⁻¹ level. The relative standard deviation at 5 × 10⁻8 g mL⁻¹ was 1.7% (n = 9). The sensitive method was successfully applied to the direct determination of lincomycin hydrochloride (ng mL⁻¹) in serum. A possible mechanism of the lumonol-DPC CL reaction was discussed by the study of the CL kinetic characteristics and the spectra of CL reaction. The oxidability of DPC was studied by means of its electrochemical response.

Effects of altered plasma alpha-1-acid glycoprotein levels on pharmacokinetics of some basic antibiotics in pigs: simulation analysis.

Effects of altered plasma alpha-1-acid glycoprotein (AGP) levels on pharmacokinetic parameters of basic antimicrobials, erythromycin (EM), lincomycin (LM) and clindamycin (CM) were evaluated in pigs by simulation analysis. Intravenous (i.v.) injections of EM, LM and CM were performed to obtain pharmacokinetic parameters in healthy conditions. Binding parameters were obtained from an in vitro study using ultrafiltration. Simulation studies indicated that an increase of plasma AGP levels resulted in a decrease of both volume of distribution at steady state (Vdss) and total body clearance (Cltot) for all the drugs. Elimination rate constant for LM was almost unchanged by an increase of plasma AGP levels, whereas those for EM and CM were increased. Plasma concentration-time profiles at a high AGP level (often observed in pathophysiological conditions) were also simulated. All of the total plasma concentration-time profiles were different from those at normal AGP level. The differences were characterized by a higher initial concentration with faster or similar elimination. Unbound plasma concentration-time profile of LM was unaffected by AGP levels, whereas EM and CM were eliminated from plasma more rapidly at high AGP level. These results suggested that adjustment of dosage regimen of EM and CM is required in pathophysiological conditions, but that of LM is not required.

[Prolonged regional intra-arterial therapy in multimodal treatment of patients with severe skeletal trauma]. / Dlitel'naia regionarnaia vnutriarterial'naia terapiia v komplekse lecheniia postradavshikh s tiazheloi skeletnoi travmoi.

A total of 108 victims with open fractures of long tubular bones of different localization are examined. Regional intraarterial therapy was added to their treatment protocols. Stable functional disorders in local hemodynamics in these patients impede the repair processes and can lead to development of infectious complications. Regional intraarterial therapy allows early elimination of hemodynamic disorders, selective antibiotic therapy, and improves the adaptation potential of the immune system.

Contribution of alpha 1-acid glycoprotein to species difference in lincosamides-plasma protein binding kinetics.

Protein binding kinetics of lincomycin (LM) and clindamycin (CM) were studied using plasma, albumin and alpha 1-acid glycoprotein (AGP) derived from humans, dogs, cattle and sheep. Based on Rosenthal plots of LM and CM, drug-binding property in plasma presented specific and non-specific binding, except for LM in cattle and sheep and for CM in sheep, where only non-specific binding was demonstrated. Dissociation constant (Kd) and binding capacity (Bmax) for specific binding and proportionality constant (PC) for non-specific binding were as follows: Kd = 3.14 mumol/L, Bmax = 15.28 mumol/L, PC = 0.19 for humans; Kd = 3.84 mumol/L, Bmax = 6.55 mumol/L, PC = 0.14 for dogs; PC = 0.12 for cattle; PC = 0.16 for sheep in LM and Kd = 0.94 mumol/L, Bmax = 12.24 mumol/L, PC = 4.98 for humans; Kd = 1.48 mumol/L, Bmax = 9.52 mumol/L, PC = 2.91 for dogs; Kd = 1.22 mumol/L, Bmax = 4.45 mumol/L, PC = 2.40 for cattle; PC = 1.48 for sheep in CM. The specific binding for each species was different, showing more difference in Bmax compared with Kd. The non-specific binding of LM was similar among species whereas that of CM was different, implying species difference. The drug-binding property of AGP for each species was all specific binding and the Kd was comparable to that obtained from plasma, indicating that AGP is a major specific binder in plasma. The lack of detection of specific binding for LM in cattle and sheep and for CM in sheep plasma could be attributable to a higher Kd and lower plasma AGP concentration compared with other species. The drug-binding property of albumin was characterized as all non-specific, without a great difference among species. Except for CM in sheep, the lower PC in albumin solution compared with that in plasma suggested the presence of another non-specific binder in plasma, i.e. lipoprotein. From the simulation of drug-binding percentage to AGP concentrations, AGP could be a major contributor to drug-plasma protein binding in pathological states. The degree of AGP-drug binding for each species could vary according to the degree of increase of AGP concentrations from a healthy to a pathological state, inducing a decrease in the unbound fraction (fp): 6.1 fold for dogs, 4.6 fold for humans, 1.8 fold for sheep and 1.4 fold for cattle in LM; 5.8 fold for dogs, 5.7 fold for cattle, 4.0 fold for humans and 1.5 fold for sheep in CM. Therefore, the disposition and efficacy of lincosamides affected by fp can be modified differently by the change of fp attributable to the alteration of plasma AGP concentration in each species.

Contribution of alpha 1-acid glycoprotein to plasma protein binding of some basic antimicrobials in pigs.

Protein binding kinetics of basic antimicrobials including trimethoprim (TMP), erythromycin (EM), lincomycin (LM) and clindamycin (CM) were studied using porcine plasma, albumin and alpha 1-acid glycoprotein (AGP). Rosenthal plots of these basic drugs in porcine plasma suggest saturable and non-saturable binding. Dissociation constants (kd) and binding capacity (Bmax) for saturable binding were as follows: TMP, kd = 8.58 mumol/L, Bmax = 5.26 mumol/L; EM, kd = 2.72 mumol/L, Bmax = 3.06 mumol/L, LM, kd = 3.96 mumol/L, Bmax = 6.58 mumol/L and CM, kd = 4.43 mumol/L, Bmax = 21.7 mumol/L. The proportionality constants (Bmax2/kd2) for non-saturable binding were 0.29 in TMP, 0.52 in EM, 0.17 in LM and 3.2 in CM. The kds of the drugs in porcine AGP solution were determined by a fluorescence quenching method, using 1-anilino-8-naphthalene sulphonate (ANS) as a fluorescent probe: 9.51 mumol/L in TMP, 1.89 mumol/L in EM, 4.48 mumol/L in LM and 9.69 mumol/L, in CM. Comparable kd values between porcine plasma and AGP solution indicated that AGP is a major saturable binder in porcine plasma. Binding property to porcine albumin presented linearity, showing the following proportionality constants: 0.23 in TMP, 0.38 in EM, 0.01 in LM and 0.76 in CM. The comparable proportionality constants of TMP and EM between porcine plasma and albumin solution indicate that albumin is a major non-saturable binder, whereas proportionality constants of LM and CM in albumin solution compared to those in porcine plasma were low, implying another non-saturable binder, i.e. lipoprotein. Simulation curve of drug-binding percentage vs. AGP concentrations showed that in pigs under a pathologic state, or during early growth stage with high AGP levels, AGP could be a main contributor to drug-plasma protein binding for all drugs examined. The increase of AGP from normal to pathological concentrations induced a decrease in the unbound fraction: LM > CM > EM > TMP in order of AGP contribution to drug binding. Therefore, the disposition and efficacy of basic antimicrobials which bind to AGP with high affinity could be markedly influenced by altered AGP levels, implying AGP contribution to pharmacokinetics and pharmacodynamics.

A reliable two-hour Staphylococcus plate assay of rifampicin, erythromycin, lincomycin, chloramphenicol and amikacin in human sera.

A simple and precise agar diffusion microassay was developed for rifampicin (RIF) in serum. The method involved the addition of sera containing RIF (0.125-4.0 micrograms/ml) to wells cut in an agar medium (pH 6.6) surface seeded with an adjusted inoculum of Staphylococcus aureus ATCC 6538 P and incubated for 2h at 40 degrees C. Repetitive assays of sera containing known concentrations of RIF revealed an average mean recovery of 93.6% with a coefficient of variation of 5.7%. This rapid method allowed accurate determination of RIF in the presence of erythromycin, chloramphenicol, lincomycin and amikacin. These four antibiotics were also separately measured in sera by the rapid assay at different pH values.

Antibiotics in exudate from periapical lesions in dogs.

Forty-eight periapical lesions were induced in the mandible of dogs. Subsequently, the dogs were given a single intramuscular injection of either benzylpenicillin, erythromycin, lincomycin or clindamycin. The antibiotic concentration in serum and exudate from the periapical lesions was measured. The highest concentration of benzylpenicillin in serum (4.3 micrograms/ml) was obtained 30 min after the injection and in exudate (1.8 micrograms/ml) 60 min after injection. The highest concentration of erythromycin in serum (0.8 microgram/ml) was reached after 120 min and in exudate (0.6 microgram/ml) 240 min after the injection. The highest concentration of clindamycin in serum (2.3 micrograms/ml) was obtained 120 min after the injection and in the exudate (2.2 micrograms/ml) after 240 min. The highest concentration of lincomycin in serum (4.2 micrograms/ml) was noted 120 min after the injection and in exudate (4.5 micrograms/ml) 240 min after the injection. The results of this study indicated that the sampling method used might be suitable for analyzing the concentration of antibiotics in a clinical situation.

Níveis sericos e tissulares (amigdalianos) de lincomicina em seguida a administraçäo de dose única de 1.2g IM/24h

Foram estudados, dois grupos de pacientes adultos que receberam doses únicas (1.2g) de Licomicina. Um grupo de 7 pacientes foi amigdalectomizado, de 10 a 12 horas após a aplicaçäo e outro grupo de 13 pacientes, após 22 a 24 horas. Em ambos grupos os níveis tissulares da droga foram dosados. Os níveis séricos foram medidos em ambos os grupos, com: 0, 2, 3, 8, 12 e 24 horas. Os resultados säo apresentados. Embora no grupo de 12 horas os níveis tissulares amigdalianos tenham sido maiores, os níveis obtidos no grupo de 24 horas revelaram-se eficientes do ponto de vista terapêutico para Streptococcus (pyogenes e pneumoniae)

Chloramphenicol, lincomycin and oxytetracycline disposition in calves with experimental pneumonic pasteurellosis.

The effects of pneumonia on the pharmacokinetics of chloramphenicol, lincomycin, and oxytetracycline were evaluated in two-month-old calves. Pneumonia was induced by injection of Pasteurella haemolytica cultures directly through the thoracic wall into each lung. Six days prior to induction of pneumonia, the antibiotics were administered in a single i.v. dose. The antibiotics were administered again 48 (i.v.), 60 and 72 h (i.m.) following injection of P. haemolytica. The pharmacokinetics of chloramphenicol (25 mg/kg) and lincomycin (10 mg/kg) were not significantly different in calves with pneumonia. The hybrid rate constant beta for oxytetracycline was increased in calves with pneumonia from 0.0034 +/- 0.0003/min to 0.0048 +/- 0.0007/min between 2 h and 8 h. Thus the elimination half-life in serum was shortened from 212.4 +/- 20.3 min to 149.3 +/- 19.5 min. In addition, there was an apparent but not statistically significant decrease in K12 with pneumonia. These findings accentuate the need for observance of 12-h dose intervals with oxytetracycline.

Protein binding and pharmacokinetics of lincomycin following intravenous administration of high doses.

High-dose infusions of lincomycin 600, 1,200, and 2,400 mg were administered to 14 healthy, adult men. Using model-independent pharmacokinetics, it was found that the half-life, mean residence time, and steady-state volume of distribution of total drug increased with dose, whereas the same parameters remained unchanged for the unbound lincomycin. Although the mean clearance value for total drug increased, this change fell short of being significant at the 5% level and was associated with a decrease in unbound clearance following administration of the 2,400 mg dose. Protein binding studies using ultrafiltration gave direct evidence of saturable serum protein binding and indicated that binding involved at least two distinct classes of binding sites.

Lincomycin serum and saliva concentrations after intramuscular injection of high doses.

Serum and saliva levels of lincomycin were determined in 15 normal adult male volunteers after a single intramuscular injection of 600, 1000, or 1500 mg lincomycin in a three-way crossover study. The area under the serum concentration-time curve did not increase in proportion to the increase in dose. The increase in concentration of lincomycin in saliva was greater than expected for the increase in dose. There were not significant differences in elimination half-life between doses. These results suggest that the apparent clearance of lincomycin increases with increasing dose possibly because of a decrease in serum protein binding of lincomycin at high serum concentrations.