During the period from April 2017 to March 2020, 44 patients were enrolled in this study and their clinical data were collected from the medical records in Affiliated Hospital of Shaoxing University, Shaoxing, Zhejiang Province, China. All lung cancer patients were diagnosed by pathological and cytological tests. Patients with severe metabolic diseases or other cancers were excluded in the study. Tracheal examination was performed in accordance with routine preoperative requirements and BALF samples were collected in clinical examinations. Sample transfer, centrifugation, and separation were completed within 3 h to avoid any preanalytical factors that might affect amino acid and carnitine concentrations. Samples were stored at − 80 °C until analysis.

The succinylacetone, non-derived amino acid, and carnitine assay kit (NZP108) was purchased from Guangzhou Fenghua Bioengineering Co., Ltd. (Guangzhou, China). This assay kit was mainly used for diagnosing metabolic diseases in clinic and the PR China Medical Device Registration Certificate Number was 2016-3-40-1324.

The detected amino acids included alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), cysteine (Cys), glutamic acid(Glu), glutamine (Gln), glycine (Gly), homocysteine (Hcy), histidine (His), leucine (Leu), lysine (Lys), methionine (Met), ornithine (Orn), phenylalanine (Phe), piperine (Pip), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val); the detected carnitines included free carnitine (C0), ethane-acyl-carnitine (C2), propane-acyl-carnitine (C3), propane-diacyl-carnitine (C3DC), butane-acyl-carnitine (C4), trihydroxy-butane-acyl-carnitine (C4OH), butane-diacyl-carnitine (C4DC), isopentane-acyl-carnitine (C5), trihydroxy-isopentane-acyl-carnitine (C5OH), pentane-diacyl-carnitine (C5DC), isopentene-acyl-carnitine (C5:1), hexane-acyl-carnitine (C6), hexane-diacyl-carnitine (C6DC), octane-acyl-carnitine (C8), decane-acyl-carnitine (C10), decene-acyl-carnitine (C10:1), decadiene-acyl-carnitine (C10:2), dodecane-acyl-carnitine (C12), tetradecane-acyl-carnitine (C14), trihydroxy-tetradecane-acyl-carnitine (C14OH), tetradecane-diacyl-carnitine(C14DC), tetradecene-acyl-carnitine (C14:1), tetradecadiene-acyl-carnitine (C14:2), hexadecane-acyl-carnitine (C16), trihydroxy-hexadecane-acyl-carnitine (C16OH), trihydroxy-hexadecene-acyl-carnitine (C16:1OH), octadecane-acyl-carnitine (C18), trihydroxy-octadecane-acyl-carnitine (C18OH), octadecene-acyl-carnitine (C18:1), trihydroxy-octadecene-acyl-carnitine (C18:1OH), octadecadiene-acyl-carnitine (C18:2), eicosane-acyl-carnitine (C20), doeicosane-acyl-carnitine (C22), tetraeicosane-acyl-carnitine (C24), and hexaeicosane-acyl-carnitine (C26); the metabolite ratios included Arg/Orn, Cit/Arg, Gly/Ala, Met/Leu, Met/Phe, Orn/Cit, Phe/Tyr, Tyr/Cit, Val/Phe, C2/C0, C3/C0, C3/C2, C3/C16, C4/C2, C4/C3, C4/C8, C5/C0, C5/C2, C5/C3, C5-OH/C8, C5-OH/C0, C5DC/C5-OH, C5DC/C16, C8/C2, C8/C10, C16-OH/C16, C26/C20, C14:1/C16, C3DC/C10, C10:2/C10, C5DC/C8, (C0 + C2 + C3 + C16 + C18:1)/Cit, (C16 + C18)/C0, C0/(C16 + C18), and C3/Met; HPLC-grade methanol was purchased from Merck (Darmstadt, Germany); AR-grade trichloromethane was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China).

One hundred microliters of BALF sample was added to 400 μL deproteinizing solution (methanol: trichloromethane, 9:1) with a mixed internal standard (amino acid and carnitine isotopic internal standard products from the assay kit). The mixture was vortexed for 1 min and then centrifuged at 13,000 rpm at 4 °C for 10 min. The 300 μl supernatant of mixture was lyophilized and then it was redissolved in 100ul extraction liquid (containing methanol and water from the assay kit). It was completely dissolved by ultrasonic at 45 ℃ for 45 min and 10 μL was used for LC–MS/MS analysis.

Amino acid and carnitine concentrations were performed by the Waters Acquity UPLC I-Class/Xevo TQD system (Waters, USA) and the LC–MS/MS detecting procedure followed the assay kit instruction. The assay kit offered the customized mobile phase and flow rate was 0.2 mL/min. The multiple reaction monitoring (MRM) and neutral loss scanning pattern were performed by mass spectrometry without UPLC separation, referring to the assay kit condition.

During detecting process, there were 2 blank samples, 2 low concentration QC samples, and 2 high concentration QC samples in the 96-well plate, and the rest were the BALF samples. Because the platform was also used for a large amount of clinical blood samples with the same assay kit, all quality control sample data on that day were listed (Table S1).

According to the QC requirement from this assay kit, the recovery of various amino acids and carnitines with isotopic internal standard products must be 80–120%, and variable coefficient of QC sample concentration must be less than 20%. When the concentration value of high or low concentration QC samples conforms to the standard value ± 3 standard deviation, the concentration values of BALF sample are considered to be effective; otherwise, they need to be detected again.

Using SPSS20.0 (IBM, USA) in the study was for basic data analysis. The Shapiro–Wilk test was used to test for normality, then the Mann–Whitney U test was performed for between group comparisons, in cases of two groups of continuous variables. And the Bonferroni correction was used to counteract the problem of multiple comparisons. The curves of receiver operating characteristic (ROC) were obtained the clinical diagnosis value of metabolites. A multivariate data analysis of PLS-DA was performed using SIMCA-P13.0 (Umetrics, Sweden).